Compositions and methods for modification of biomolecules

ABSTRACT

The present invention provides modified cycloalkyne compounds; and method of use of such compounds in modifying biomolecules. The present invention features a cycloaddition reaction that can be carried out under physiological conditions. In general, the invention involves reacting a modified cycloalkyne with an azide moiety on a target biomolecule, generating a covalently modified biomolecule. The selectivity of the reaction and its compatibility with aqueous environments provide for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

CROSS-REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/264,463, filed Oct. 31, 2005, which claims the benefit ofU.S. Provisional Patent Application No. 60/624,202 filed Nov. 1, 2004,which applications are incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant numberGM058867 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention relates generally to covalent modification of moleculesuseful in, for example, modification of surfaces (including cellsurfaces), and modification of molecules under physiological conditions(e.g., in a cellular environment).

BACKGROUND OF THE INVENTION

Selective chemical reactions that are orthogonal to the diversefunctionality of biological systems are now recognized as importanttools in chemical biology. As relative newcomers to the repertoire ofsynthetic chemistry, these bioorthogonal reactions have inspired newstrategies for compound library synthesis, protein engineering,functional proteomics, and chemical remodeling of cell surfaces. Theazide has secured a prominent role as a unique chemical handle forbioconjugation. The Staudinger ligation has been used with phosphines totag azidosugars metabolically introduced into cellular glycoconjugates.The Staudinger ligation can be performed in living animals withoutphysiological harm; nevertheless, the Staudinger reaction is not withoutliabilities. The requisite phosphines are susceptible to air oxidationand their optimization for improved water solubility and increasedreaction rate has proven to be synthetically challenging.

The azide group has an alternative mode of bioorthogonal reactivity: the[3+2] cycloaddition with alkynes described by Huisgen. In its classicform, this reaction has limited applicability in biological systems dueto the requirement of elevated temperatures (or pressures) forreasonable reaction rates. Sharpless and coworkers surmounted thisobstacle with the development of a copper(I)-catalyzed version, termed“click chemistry,” that proceeds readily at physiological temperaturesand in richly functionalized biological environs. This discovery hasenabled the selective modification of virus particles, nucleic acids,and proteins from complex tissue lysates. Unfortunately, the mandatorycopper catalyst is toxic to both bacterial and mammalian cells, thusprecluding applications wherein the cells must remain viable.Catalyst-free Huisgen cycloadditions of alkynes activated byelectron-withdrawing substituents have been reported to occur at ambienttemperatures. However, these compounds undergo Michael reaction withbiological nucleophiles.

There is a need in the field for additional mechanisms to modifybiological molecules through a biocompatible reaction, particularly in abiological environment.

LITERATURE

Huisgen (1963) Angew. Chem. Int. Ed. 2:565-598; Shea and Kim. J. Am.Chem. Soc. 1992, 114, 4846-4855; Reese and Shaw (1970) Chem. Comm.1172-1173; Wilbur et al. Bioconj. Chem. 1996, 7, 689-702; Bistrup et al.J. Cell Biol. 1999, 145, 899-910; Saxon et al. J. Am. Chem. Soc. 2002,124, 14893-14902; Hang and Bertozzi. Acc. Chem. Res. 2001, 34, 727-736;Link et al. Curr. Opin. Biotechnol. 2003, 14, 603-609; Lee et al. J. Am.Chem. Soc. 2003, 125, 9588-9589; Wang et al. J. Am. Chem. Soc, 2003,125, 3192-3193; Kiick et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,19-24; Speers and Cravatt. Chem. Biol. 2004, 11, 535-546; Saxon andBertozzi Science 2000, 287, 2007-2010; Link, A. J.; Tirrell, D. A. J.Am. Chem. Soc. 2003, 125, 1164-1165; Dube and Bertozzi. Curr. Opin.Chem. Biol. 2003, 7, 616-625; Vocadlo et al. Proc. Natl. Acad. Sci.U.S.A. 2003, 100, 9116-9121; Prescher et al. Nature 2004, 430, 873-877;Seo et al. J. Org. Chem. 2003, 68, 609-612; Li et al. Tetrahedron Lett.2004, 45, 3143-3146; Wittig and Krebs. Chem. Ber. 1961, 94, 3260-3275;Meier et al. Chem. Ber. 1980, 113, 2398-2409; Turner et al. J. Am. Chem.Soc. 1972, 95, 790-792.

SUMMARY OF THE INVENTION

The present invention provides modified cycloalkyne compounds; andmethod of use of such compounds in modifying biomolecules. The presentinvention features a cycloaddition reaction that can be carried outunder physiological conditions. In general, the method involves reactinga modified cycloalkyne with an azide moiety on a target biomolecule,generating a covalently modified biomolecule. The selectivity of thereaction and its compatibility with aqueous environments provide for itsapplication in vivo (e.g., on the cell surface or intracellularly) andin vitro (e.g., synthesis of peptides and other polymers, production ofmodified (e.g., labeled) amino acids).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict cycloaddition reactions. FIG. 1A depictsCu(I)-catalyzed Huisgen cycloaddition (“click chemistry”). FIG. 1Bdepicts strain-promoted [3+2] cycloaddition of azides and cyclooctynes.

FIG. 2 depicts labeling of azide-modified GlyCAM-Ig with alkyne probes.

FIGS. 3A-C depict cell surface labeling with a modified cyclooctynecompound.

FIG. 4A schematically depicts labeling of cell-surface azides withcyclooctyne probes.

FIG. 4B depicts cyclooctyne probe labeling of Jurkat cells that containcell-surface azides.

FIG. 5 depicts labeling of splenocytes with cyclooctyne-FLAG.

FIG. 6 depicts in vivo labeling of mice with cyclooctyne-FLAG compounds.

FIG. 7 presents a Western blot of Jurkat cells lysate treated with (⁺Az)or without (⁻Az) Ac₄ManNAz and labeled with 0 or with 250 μMazacycloocytne biotin conjugate 9.

FIG. 8 presents data showing cell surface labeling of Jurkat cells grownin the presence (Az) or absence (no Az) of Ac₄ManNAz for 3 days.

FIG. 9 depicts in vivo reaction of DIFO-FLAG with metabolically labeledazido glycans.

FIG. 10 depicts time-dependent labeling of an isolated azidoprotein byCu-catalyzed (lower panedl) or Cu-free (DIFO; upper panel) clickchemistry, using Alexa Fluor 488 derivatives.

FIGS. 11A and 11B depict cell surface labeling of azido glycans byvarious biotinylated derivatives of cyclooctynes or phosphines.

FIG. 12 presents comparative cell surface labeling of azido glycans bybiotinylated derivatives of DIFO, DIFO2, and DIFO3.

FIG. 13 depicts concentration dependence of the reaction of DIFO2-biotinand DIFO3-biotine with cell surface azido glycans.

DEFINITIONS

By “reactive partner” is meant a molecule or molecular moiety thatspecifically reacts with another reactive partner. Exemplary reactivepartners are those of the reaction of the invention, i.e., an azidegroup of an azide-modified target molecule and the cycloalkyne group ofa modified cycloalkyne moiety.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment or its syntheticenvironment and is at least 60% free, at least 75% free, at least 90%free, at least 95% free, at least 98% free, or at least 99% free fromother components with which it is naturally associated, or is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, or at least 99% free from contaminants associated withsynthesis of the compound.

As used herein, the term “cell” in the context of the in vivoapplications of the invention is meant to encompass eukaryotic andprokaryotic cells of any genus or species, with mammalian cells being ofparticular interest. “Cell” is also meant to encompass both normal cellsand diseased cells, e.g., cancerous cells. In many embodiments, thecells are living cells.

The terms “polypeptide” and “protein,” used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

The term “aryl” as used herein means 5- and 6-membered single-aromaticradicals which may include from zero to four heteroatoms. Representativearyls include phenyl, thienyl, furanyl, pyridinyl, (is)oxazoyl and thelike.

The term “lower alkyl”, alone or in combination, generally means anacyclic alkyl radical containing from 1 to about 10, e.g., from 1 toabout 8 carbon atoms, or from 1 to about 6 carbon atoms. Examples ofsuch radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl and thelike.

The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, trifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy,C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy, oxycarboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, carboxamide, protectedcarboxamide, N—(C₁ to C₆ alkyl)carboxamide, protected N—(C₁ to C₆alkyl)carboxamide, N,N-di(C₁ to C₆ alkyl)carboxamide, trifluoromethyl,N—((C₁ to C₆ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,substituted or unsubstituted, such that, for example, a biphenyl ornaphthyl group results.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six memberedcarbocyclic rings. Also as noted above, the term “heteroaryl” denotesoptionally substituted five-membered or six-membered rings that have 1to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, e.g., to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl. Substituents for the heteroaryl group are asheretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C₁ to C₄ alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ toC₇ substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature, or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or di-substituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amodified cycloalkyne” includes a plurality of such modified cycloalkynesand reference to “the target molecule” includes reference to one or moretarget molecules and equivalents thereof known to those skilled in theart, and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features a strain-promoted [3+2] cycloadditionreaction that can be carried out under physiological conditions. Ingeneral, the invention involves reacting a modified cycloalkyne with anazide moiety on a biomolecule, generating a covalently modifiedbiomolecule. The selectivity of the reaction and its compatibility withaqueous environments provides for its application in vivo (e.g., on thecell surface or intracellularly) and in vitro (e.g., synthesis ofpeptides and other polymers, production of modified (e.g., labeled)amino acids). The reaction is compatible with modification of livingcells.

The invention provides methods and compositions for specifically andefficiently synthetically modifying cellular components in an aqueousenvironment, thus providing for modification of such cellular componentson or in living cells. The invention uses reactive partners that arecompletely abiotic and are chemically orthogonal to native cellularcomponents, thus providing for extreme selectivity of the reaction.Furthermore, the reaction can be carried out under physiologicalconditions, e.g., a pH of about 7 within an aqueous environment, and atabout 37° C.

The invention is based in part on the discovery of a means for carryingout a modified Huisgen reaction that can be carried out in an aqueous,physiological environment. Because the reaction of the invention ishighly selective and functions in aqueous solvents, the reaction can beused in a variety of applications both in vitro and in vivo. Thereaction is accomplished through use of a first molecule comprising astrained cycloalkyne moiety, and second molecule comprising an azidemoiety. The azide moiety on the second molecule reacts, in the absenceof a catalyst, with the strained cycloalkyne moiety on the firstmolecule, forming a final conjugate product comprising fusedazide/cycloalkyne ring. The first molecule comprising the strainedcycloalkyne moiety can further comprise a moiety that allows forsubsequent reactions and/or which provides for detectable labeling ofthe product of the final reaction. The reaction proceeds without theneed for a catalyst. Instead, activation energy for the reaction isprovided by azide group and the strained cycloalkyne group. Theinvention takes advantage of the massive bond angle deformation of theacetylene group in the cycloalkyne moiety, which provides for ringstrain. For example, the bond angle deformation of the acetylene groupof cyclooctyne to 163° accounts for nearly 18 kcal/mol of ring strain.This destabilization of the ground state versus the transition state ofthe reaction provides a dramatic rate acceleration compared tounstrained alkynes.

Modified Cycloalkyne Compounds

The present invention provides modified cycloalkyne compounds; andcompositions comprising the compounds. A subject modified cycloalkyne isa compound of the formula:Y—R₁—X

where:

X is a strained cycloalkyne group, or a heterocycloalkyne group,substituted with R₁, and in some embodiments one or more additionalgroups;

Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest; and

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. Insome embodiments, R₁ is a substituted or unsubstituted aliphatic group,e.g., a substituted or unsubstituted alkyl; a substituted orunsubstituted alkenyl; or a substituted or unsubstituted alkynyl. Insome embodiments, R₁ is a substituted or unsubstituted phenyl.

In some embodiments, Y is a reactive group. Suitable reactive groupsinclude, but are not necessarily limited to, carboxyl, amine, (e.g.,alkyl amine (e.g., lower alkyl amine), aryl amine), ester (e.g., alkylester (e.g., lower alkyl ester, benzyl ester), aryl ester, substitutedaryl ester), thioester, sulfonyl halide, alcohol, thiol, succinimidylester, isothiocyanate, iodoacetamide, maleimide, hydrazine, and thelike. In some embodiments, Y is a reactive group selected from acarboxyl, an amine, an ester, a thioester, a sulfonyl halide, analcohol, a thiol, a succinimidyl ester, an isothiocyanate, aniodoacetamide, a maleimide, and a hydrazine.

In some embodiments, Y is a molecule of interest, where suitablemolecules of interest include, but are not limited to, a detectablelabel; a toxin (including cytotoxins); a linker; a peptide; a drug; amember of a specific binding pair; an epitope tag; and the like. Where Yis a molecule of interest other than a linker, the molecule of interestis attached directly to R₁, or is attached through a linker.

The cycloalkyne is a strained cycloalkyne, e.g., the cycloalkyneincreases the rate of reaction from about 2-fold to about 1000-fold,e.g., the cycloalkyne increases the rate of reaction at least about2-fold, at least about 5-fold, at least about 10-fold, at least about50-fold, at least about 100-fold, at least about 500-fold, or at leastabout 1000-fold, compared to the rate of reaction between an azide and alinear alkyne having the same number of carbon atoms as the cycloalkyne.The strained cycloalkyne will in some embodiments be aheterocycloalkyne, e.g., the cycloalkyne will in some embodimentscomprise atoms other than carbon. In some embodiments, the cycloalkyneor heterocycloalkyne will be a 7-membered ring. In other embodiments,the cycloalkyne or heterocycloalkyne will be an 8-membered ring. Inother embodiments, the cycloalkyne or heterocycloalkyne will be a9-membered ring. The strain on the cycloalkyne can be increased in avariety of ways, e.g., through the use of heteroatoms; the degree ofunsaturation, or torsional strain; the use of electron-withdrawinggroups, etc. In some embodiments of particular interest, the cycloalkyneis a cyclooctyne. In some embodiments, the strained cycloalkyne is acompound in which one or more of the carbon atoms in the cycloalkynering, other than the two carbon atoms joined by a triple bond, issubstituted with one or more electron-withdrawing groups, e.g., a halo(bromo, chloro, fluoro, iodo), a nitro group, a cyano group, or asulfone group. Where the electron-withdrawing group is a nitro group, acyano group, or a sulfone group, the electron-withdrawing group is notdirectly linked to the cycloalkyne ring.

In some embodiments, a subject modified cycloalkyne is of Formula I:

where:

Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest; and

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁can be at any position on the cyclooctyne group other than at the twocarbons joined by the triple bond.

Exemplary, non-limiting examples of a subject cyclooctyne compound,e.g., an exemplary cyclooctyne compound of Formula I include:

In some embodiments, the modified cycloalkyne is of Formula I, whereinone or more of the carbon atoms in the cyclooctyne ring, other than thetwo carbon atoms joined by a triple bond, is substituted with one ormore electron-withdrawing groups, e.g., a halo (bromo, chloro, fluoro,iodo), a nitro group, a cyano group, a sulfone group, or a sulfonic acidgroup. Thus, e.g., in some embodiments, a subject modified cycloalkyneis of Formula II:

where:

each of X and X′ is independently:

(a) H;

(b) one or two halogen atoms (e.g., bromo, chloro, fluoro, iodo);

(c) —W—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or4); W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid,or a halogen);

(d) —(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or2; W is O, N, S, or sulfonyl; if W is O, N, or S, then Z is nitro,cyano, or halogen; and if W is sulfonyl, then Z is H); or

(e) —(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or4); and Z is nitro, cyano, sulfonic acid, or a halogen);

Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest; and

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁can be at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond.

Exemplary, non-limiting examples of a subject cyclooctyne compound,e.g., an exemplary cyclooctyne compound of Formula II, include:

In some embodiments, a subject modified cycloalkyne is of Formula III:

wherein each of R₁-R₆ is independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; a nitro; —W—(CH₂)_(n)—Z (where: n is an integerfrom 1-4 (e.g., n=1, 2, 3, or 4), wherein W, if present, is O, N, or S;and Z is nitro, cyano, sulfonic acid, or a halogen);—(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or 2;W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro,cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or 4),and wherein Z is nitro, cyano, sulfonic acid, or a halogen);

wherein R₃ is optionally linked to R through Y thus forming asubstituted or unsubstituted cycloalkyl or substituted or unsubstitutedheterocycloalkyl substituent on the cycloalkyne ring, wherein Y, ifpresent, is C, O, N, or S; and

wherein each of R₁-R₆ is optionally independently linked to a moietythat comprises a reactive group that facilitates covalent attachment ofa molecule of interest; or a molecule of interest.

In some embodiments of Formula III, R₁ is two fluoride atoms, one ormore of R₂, R₃, R₄, and R₅ is a fluorophore, and R₆ is —OR₇, where —OR₇is a leaving group with a quencher (e.g., and ester, a sulfonate, etc.).

Exemplary, non-limiting examples of a subject halogenated cyclooctynecompound, e.g., a compound of Formula III, include:

where R₈ is selected from H; a halogen atom (e.g., bromo, fluoro,chloro, iodo); an aliphatic group, a substituted or unsubstituted alkylgroup; an alkenyl group; an alkynyl group; a carboxylic acid, an alkylester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; athiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonylester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; and a nitro.

In some embodiments, a subject modified cycloalkyne is of Formula IV:

where:

Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest; and

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁can be at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond.

Exemplary, non-limiting examples of a subject halogenated cyclooctynecompound, e.g., a compound of Formula IV, include:

In some embodiments, a subject modified cycloalkyne is of Formula V:

where:

Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest; and

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁can be at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond, and other than thefluoride-substituted carbon.

Exemplary, non-limiting examples of a subject halogenated cyclooctynecompound, e.g., exemplary compounds of Formula V, include:

In some embodiments, a subject modified cycloalkyne is of Formula VI:

where Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest; and

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁can be at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond, and other than thefluoride-substituted carbons.

In some embodiments, a subject modified cycloalkyne is of Formula VII:

wherein five of X₁-X₆ are carbon atoms;

wherein one of X₁-X₆ is N, O, P, or S;

wherein each of R₁-R₆ is independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; a nitro; —W—(CH₂)_(n)—Z (where: n is an integerfrom 1-4 (e.g., n=1, 2, 3, or 4), wherein W, if present, is O, N, or S;and Z is nitro, cyano, sulfonic acid, or a halogen);—(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or 2;W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro,cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or 4),and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and

wherein each of R₁-R₆ is optionally independently linked to a moietythat comprises a reactive group that facilitates covalent attachment ofa molecule of interest; or a molecule of interest.

In some embodiments, a subject compound is a compound of one of FormulasVIII and IX:

where each —OR is independently a leaving group.

In some embodiments, a subject modified cycloalkyne is a heteroalkyne ofFormula X:

wherein each of R₁-R₆ is independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; a methoxy group;an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; anamine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; asulfonyl ester; an alkyl ketone; an aryl ketone; a substituted arylketone; a halosulfonyl; a nitrile; a nitro; —W—(CH₂)_(n)—Z (where: n isan integer from 1-4 (e.g., n=1, 2, 3, or 4), wherein W, if present, isO, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen);—(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or 2;W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro,cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or 4),and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and

wherein each of R₁-R₆ is optionally independently linked to a moietythat comprises a reactive group that facilitates covalent attachment ofa molecule of interest; or a molecule of interest.

Exemplary, non-limiting examples of a subject azacyclooctyne compound,exemplary compounds of Formula X, include:

In some embodiments, a subject compound has the structure of one ofFormulas XI-XVI:

where R, R₁, R₂, and R₃ are each independently H; one or two halogenatoms (e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; a methoxygroup; an alkyl ester; an aryl ester; a substituted aryl ester; analdehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; athioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; asubstituted aryl ketone; a halosulfonyl; a nitrile; or a nitro;

wherein —OR is in some embodiments a leaving group with a quencher; and

wherein each R is optionally independently linked to a moiety thatcomprises a reactive group that facilitates covalent attachment of amolecule of interest; or a molecule of interest.

In some embodiments, a subject compound has the structure of FormulaXVII:

where R₁ and R₂ are each independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; or a nitro; and

wherein each of R₁ and R₂ is optionally independently linked to a moietythat comprises a reactive group that facilitates covalent attachment ofa molecule of interest; or a molecule of interest.

Exemplary, non-limiting examples of a subject azacyclooctyne compound,exemplary compounds of Formula XVII, include:

In some embodiments, a subject compound has the structure of FormulaXVIII:

where Z is —CH₂—CH₂—, —CH═CH—, —CH≡CH—, —Se(O)O—, —C(O)O—, —C(R₃)(R₄)O—,—N(R₅)N(R₆)—, —CH(OR₇)CH₂—, or —S(O)O—;

where R₁ and R₂ are each independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; or a nitro;

where R₃ to R₇ is each independently selected from H; a halogen atom(e.g., bromo, fluoro, chloro, iodo); an aliphatic group, a substitutedor unsubstituted alkyl group; an alkenyl group; an alkynyl group; acarboxylic acid, an alkyl ester; an aryl ester; a substituted arylester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkylhalide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone;a substituted aryl ketone; a halosulfonyl; a nitrile; or a nitro; and

wherein each of R₁ and R₂ is optionally independently linked to a moietythat comprises a reactive group that facilitates covalent attachment ofa molecule of interest; or a molecule of interest.

In some embodiments, a subject compound has the structure of one ofFormulas XIX, XX, and XXI:

where R₁ and R₂ are as defined above for Formula XVIII.

In some embodiments, a subject compound has the structure of FormulaXXII:

wherein are each independently H; one or two halogen atoms (e.g., bromo,chloro, fluoro, iodo); a carboxylic acid; an alkyl ester; an aryl ester;a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; anaryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkylketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; anitrile; or a nitro;

wherein each n is independently 0, 1, or 2; and

wherein each of R₁ and R₂ is optionally independently linked to a moietythat comprises a reactive group that facilitates covalent attachment ofa molecule of interest; or a molecule of interest.

Molecules of Interest

In some embodiments, Y is a molecule of interest. Suitable molecules ofinterest include, but are not limited to, a detectable label; a toxin(including cytotoxins); a linker; a peptide; a drug; a member of aspecific binding pair; an epitope tag; and the like. Where Y is amolecule of interest other than a linker, the molecule of interest isattached directly to an R group, as noted above, or is attached througha linker.

Where Y is a molecule of interest, the modified cycloalkyne comprises amolecule desired for delivery and conjugation to the azido-targetsubstrate (azide-containing target molecule), which target substrate maybe displayed on the cell surface, may reside within the cell membrane,or may be intracellular. Molecules that may be desirable for deliveryinclude, but are not necessarily limited to, detectable labels (e.g.,spin labels, fluorescence resonance energy transfer (FRET)-type dyes,e.g., for studying structure of biomolecules in vivo), small moleculedrugs, cytotoxic molecules (e.g., drugs), ligands for binding by atarget receptor (e.g., to facilitate viral attachment, attachment of atargeting protein present on a liposome, etc.), tags to aid inpurification by, for example, affinity chromatography (e.g., attachmentof a FLAG epitope), and molecules to facilitate selective attachment ofthe polypeptide to a surface, and the like. Specific, non-limitingexamples are provided below.

Detectable Labels

The compositions and methods of the invention can be used to deliver adetectable label to a target molecule having an azide. Thus, in someembodiments, a subject modified cycloalkyne comprises a detectablelabel, covalently bound to the modified cycloalkyne either directly orthrough a linker.

Exemplary detectable labels include, but are not necessarily limited to,fluorescent molecules (e.g., autofluorescent molecules, molecules thatfluoresce upon contact with a reagent, etc.), radioactive labels (e.g.,¹¹¹In, ¹²⁵I, ¹³¹I, ²¹²B, ⁹⁰Y, ¹⁸⁶Rh, and the like); biotin (e.g., to bedetected through reaction of biotin and avidin); fluorescent tags;imaging reagents (e.g., those described in U.S. Pat. No. 4,741,900 andU.S. Pat. No. 5,326,856), and the like. Detectable labels also includepeptides or polypeptides that can be detected by antibody binding, e.g.,by binding of a detectably labeled antibody or by detection of boundantibody through a sandwich-type assay. Also suitable for use arequantum dots (e.g., detectably labeled semiconductor nanocrystals, suchas fluorescently labeled quantum dots, antibody-conjugated quantum dots,and the like). See, e.g., Dubertret et al. 2002 Science 298:759-1762;Chan et al. (1998) Science 281:2016-2018; U.S. Pat. No. 6,855,551;Bruchez et al. (1998) Science 281:2013-2016

Suitable fluorescent molecules (fluorophores) include, but are notlimited to, fluorescein, fluorescein isothiocyanate, succinimidyl estersof carboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer offluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM(tetramethylrhodamine-, methyl ester), TMRE (tetramethylrhodamine, ethylester), tetramethylrosamine, rhodamine B and4-dimethylaminotetramethylrosamine, green fluorescent protein,blue-shifted green fluorescent protein, cyan-shifted green fluorescentprotein, red-shifted green fluorescent protein, yellow-shifted greenfluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-cacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)amino-1-fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl hodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, and fluorescent europium and terbium complexes;and the like. Fluorophores of interest are further described in WO01/42505 and WO 01/86001.

Suitable fluorescent proteins and chromogenic proteins include, but arenot limited to, a green fluorescent protein (GFP), including, but notlimited to, a GFP derived from Aequoria victoria or a derivativethereof, e.g., a “humanized” derivative such as Enhanced GFP, which isavailable commercially, e.g., from Clontech, Inc.; a GFP from anotherspecies such as Renilla reniformis, Renilla mulleri, or Ptilosarcusguernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J.Protein Chem. 20:507-519; “humanized” recombinant GFP (hrGFP)(Stratagene); any of a variety of fluorescent and colored proteins fromAnthozoan species, as described in, e.g., Matz et al. (1999) NatureBiotechnol. 17:969-973; and the like.

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., CYPYDVPDYA; SEQ ID NO:1), FLAG (e.g., DYKDDDDK; SEQ ID NO:2),FLAG-C (e.g., DYKDDDDKC; SEQ ID NO:3, c-myc (e.g., CEQKLISEEDL; SEQ IDNO:4), a metal ion affinity tag such as a polyhistidine tag (e.g.,His₆), and the like.

Suitable imaging agents include positive contrast agents and negativecontrast agents. Suitable positive contrast agents include, but are notlimited to, gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA);Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA);Gadolinium-1,4,7-tris(carbonylmethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazacyclododecane(Gd-HP-DO3A); Manganese(II)-dipyridoxal diphosphate (Mn-DPDP);Gd-diethylenetriaminepentaacetate-bis(methylamide) (Gd-DTPA-BMA); andthe like. Suitable negative contrast agents include, but are not limitedto, a superparamagnetic iron oxide (SPIO) imaging agent; and aperfluorocarbon, where suitable perfluorocarbons include, but are notlimited to, fluoroheptanes, fluorocycloheptanes,fluoromethylcycloheptanes, fluorohexanes, fluorocyclohexanes,fluoropentanes, fluorocyclopentanes, fluoromethylcyclopentanes,fluorodimethylcyclopentanes, fluoromethylcyclobutanes,fluorodimethylcyclobutanes, fluorotrimethylcyclobutanes, fluorobutanes,fluorocyclobutanse, fluoropropanes, fluoroethers, fluoropolyethers,fluorotriethylamines, perfluorohexanes, perfluoropentanes,perfluorobutanes, perfluoropropanes, sulfur hexafluoride, and the like.

Specific Binding Partners

In another embodiment, a subject modified cycloalkyne comprises a memberof a pair of binding partners A member of a pair of binding partners isreferred to herein as a “specific binding partner.”

Suitable specific binding partners include, but are not limited to, amember of a receptor/ligand pair; a member of an antibody/antigen pair;a member of a lectin/carbohydrate pair; a member of an enzyme/substratepair; biotin/avidin; biotin/streptavidin; digoxin/antidigoxin; and thelike. Suitable specific binding partners include, but are not limited toa receptor ligand; a receptor for a ligand; a ligand-binding portion ofa receptor; an antibody; an antigen-binding fragment of an antibody; anantigen; a hapten; a lectin; a lectin-binding carbohydrate; an enzymesubstrate; an irreversible inhibitor of an enzyme (e.g., an irreversibleinhibitor that binds a substrate binding site of an enzyme, e.g., a“suicide” substrate); and the like.

Suitable ligand members of receptor/ligand pairs include, but are notlimited to, neurotransmitters such as opioid compounds, acetylcholine,and the like; viral proteins that bind to a cell surface receptor, e.g.,human immunodeficiency virus gp120, and the like; hormones; and thelike.

Suitable antigen-binding antibody fragments include F(ab′)₂, F(ab)₂,Fab′, Fab, Fv, scFv, and Fd fragments, single-chain antibodies, andfusion proteins comprising an antigen-binding portion of an antibody anda non-antibody protein (e.g., an antigen-binding fragment of an antibodyfused to an immunoglobulin constant region).

Suitable haptens include, but are not limited to,(4-hydroxy-3-nitrophenyl)acetyl; diethylenetriaminepentaacetic acid(DTPA) or one of its metal complexes; paranitrophenyl; biotin;fluorescein isothiocyanate; and the like.

Drugs

Suitable drugs that can be attached to a modified cycloalkyne moietyinclude, but are not limited to, cytotoxic compounds (e.g., cancerchemotherapeutic compounds); antiviral compounds; biological responsemodifiers (e.g., hormones, chemokines, cytokines, interleukins, etc.);microtubule affecting agents; hormone modulators; steroidal compounds;and the like.

Suitable cancer chemotherapeutic compounds include, but are not limitedto, non-peptidic (i.e., non-proteinaceous) compounds that reduceproliferation of cancer cells; peptidic compounds that reduceproliferation of cancer cells; anti-metabolite agents; cytotoxic agents;and cytostatic agents. Non-limiting examples of chemotherapeutic agentsinclude alkylating agents, nitrosoureas, antimetabolites, antitumorantibiotics, plant (vinca) alkaloids, and steroid hormones.

Suitable agents that act to reduce cellular proliferation include, butare not limited to, alkylating agents, such as nitrogen mustards,nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes,including, but not limited to, mechlorethamine, cyclophosphamide(Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine(CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracilmustard, chlormethine, ifosfamide, chlorambucil, pipobroman,triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Suitable antimetabolite agents include folic acid analogs, pyrimidineanalogs, purine analogs, and adenosine deaminase inhibitors, including,but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside,fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine(6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable anti-proliferative natural products and their derivatives,(e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, andepipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel(Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C,L-asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine,vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g.etoposide, teniposide, etc.; antibiotics, e.g. anthracycline,daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine),idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.;phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides,e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin);anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g.mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506(tacrolimus, prograf), rapamycin, etc.; and the like.

Other suitable anti-proliferative cytotoxic agents are navelbene,CPT-11, anastrazole, letrazole, capecitabine, reloxafine,cyclophosphamide, ifosamide, and droloxafine.

Suitable microtubule affecting agents that have antiproliferativeactivity include, but are not limited to, allocolchicine (NSC 406042),Halichondrin B (NSC 609395), colchicine (NSC 757), colchicinederivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine(NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol®derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), tritylcysterin, vinblastine sulfate, vincristine sulfate, natural andsynthetic epothilones including but not limited to, eopthilone A,epothilone B, discodermolide; estramustine, nocodazole, and the like.

Suitable hormone modulators and steroids (including synthetic analogs)include, but are not limited to, adrenocorticosteroids, e.g. prednisone,dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesteronecaproate, medroxyprogesterone acetate, megestrol acetate, estradiol,clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol,testosterone, fluoxymesterone, dromostanolone propionate, testolactone,methylprednisolone, methyl-testosterone, prednisolone, triamcinolone,chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine,medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil),Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferationand differentiation, therefore compounds that bind to the estrogenreceptor are used to block this activity. Corticosteroids may inhibit Tcell proliferation.

Other suitable chemotherapeutic agents include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; andhydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomeraseinhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

Taxanes are also suitable for attachment to a cycloalkyne moiety.“Taxanes” include paclitaxel, as well as any active taxane derivative orpro-drug. “Paclitaxel” (which should be understood herein to includeanalogues, formulations, and derivatives such as, for example,docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetylanalogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253;5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267),or obtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; orT-1912 from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Biological response modifiers that are suitable for attachment to acycloalkyne moiety include, but are not limited to, (1) inhibitors oftyrosine kinase (RTK) activity; (2) inhibitors of serine/threoninekinase activity; (3) tumor-associated antigen antagonists, such asantibodies that bind specifically to a tumor antigen; (4) apoptosisreceptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ (8)colony-stimulating factors; and (9) inhibitors of angiogenesis.

Linkers

Suitable linkers include, but are not limited to, a carboxylic acid, analkyl ester, an aryl ester, a substituted aryl ester, an aldehyde, anamide, an aryl amide, an alkyl halide, a thioester, a sulfonyl ester, analkyl ketone, an aryl ketone, a substituted aryl ketone, a halosulfonyl,a nitrile, a nitro, and a peptide linker.

Exemplary peptide linkers for use in linking a molecule of interest to amodified cycloalkyne will in some embodiments have a combination ofglycine, alanine, proline and methionine residues, where suitablepeptide linkers include, but are not limited to AAAGGM (SEQ ID NO:5);AAAGGMPPAAAGGM (SEQ ID NO:6); AAAGGM (SEQ ID NO:7); and PPAAAGGMM (SEQID NO: 8). In some embodiments, a peptide linker will comprise multipleserine residues, e.g., from 50% to 75%, or from 75% to 100% of the aminoacids in the linker are serine residues. In other embodiments, a peptidelinker will comprise multiple glycine residues, e.g., from 50% to 75%,or from 75% to 100% of the amino acids in the linker are glycineresidues. Any flexible linker, generally having a length of from about 6amino acids and about 40 amino acids is suitable for use. Linkers mayhave virtually any sequence that results in a generally flexiblepeptide, including alanine-proline rich sequences.

Compositions

The present invention further provides compositions, includingpharmaceutical compositions, comprising a subject modified cycloalkynecompound. A subject composition generally comprises a subject modifiedcycloalkyne compound; and at least one additional compound. Suitableadditional compounds include, but are not limited to: a salt, such as amagnesium salt, a sodium salt, etc., e.g., NaCl, MgCl, KCl, MgSO₄, etc.;a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; and the like.

In some embodiments, a subject composition comprises a subject modifiedcycloalkyne compound; and a pharmaceutically acceptable excipient. Awide variety of pharmaceutically acceptable excipients are known in theart and need not be discussed in detail herein. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20^(th) edition, Lippincott,Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug DeliverySystems (1999) H. C. Ansel et al., eds., 7^(th) ed., Lippincott,Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^(rd) ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Methods of Modifying a Target Biomolecule

The present invention provides methods for chemoselective modificationof a target molecule comprising an azide. The methods generally involvereacting an azide in an azide-containing target molecule with a modifiedcycloalkyne. The modified cycloalkyne has a structure as describedabove. Thus, in many embodiments, a subject method for syntheticallymodifying a cellular component generally involves: a) introducing anazide moiety into a cellular component, thereby generating anazide-modified cellular component; and contacting the cell comprisingthe azide-modified cellular component with a reactive partner comprisinga modified cycloalkyne, the contacting being under physiologicalconditions. The contacting step results in reaction between the azidegroup of azide-modified cellular component and the cycloalkyne of thereactive partner, thereby synthetically and covalently modifying thecellular component. In some embodiments, the method is carried out onliving cells in vitro. In other embodiments, the method is carried outon living cells ex vivo. In still other embodiments, the method iscarried out on living cells in vivo.

In one embodiment, the chemoselective ligation is designed for use infully aqueous, physiological conditions and involves production of astable, final product comprising a fused azide/cycloalkyne ring. Ingeneral, this embodiment involves reacting a first reactant comprising astrained cycloalkyne moiety with a second reactant comprising an azide,such that a covalent bond is formed between the first and secondreactants by reaction of the strained cycloalkyne moiety with the azidegroup.

First Reactant

A first reactant comprises a strained cycloalkyne moiety that providesthe energy for the reaction between the first and second reactants. Thefirst reactant is a modified cycloalkyne compound of the formula:Y—R₁—X

where:

X is a cycloalkyne group substituted with R₁;

Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest;

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. Insome embodiments, the first reactant is a modified cycloalkyne compoundof any of Formulas I-XXII, as described above.

Exemplary reactive groups include, but are not necessarily limited to,carboxyl, amine, (e.g., alkyl amine (e.g., lower alkyl amine), arylamine), ester (e.g., alkyl ester (e.g., lower alkyl ester, benzylester), aryl ester, substituted aryl ester), thioester, sulfonyl halide,alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide,maleimide, hydrazine, and the like. Exemplary molecules of interestfurther include dyes (e.g., fluorescein or modified fluorescein, and thelike), toxins (including cytotoxins), linkers, peptides, and the like.

The molecule of interest may be reacted directly with the reactive groupor through a linker. Exemplary molecules of interest include, but arenot necessarily limited to, a detectable label, a drug, a peptide, amember of a specific binding pair, and the like. Such molecules ofinterest are described in more detail above.

In some embodiments, Y is a reactive group selected from a carboxyl, anamine, an ester, a thioester, a sulfonyl halide, an alcohol, a thiol, asuccinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide,and a hydrazine.

In some embodiments, the cycloalkyne is a cyclooctyne.

In some embodiments, the modified cycloalkyne is of Formula I:

where

Y is H; a moiety that comprises a reactive group that facilitatescovalent attachment of a molecule of interest; or a molecule ofinterest;

R₁ is selected from a carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro.

In other embodiments, the modified cycloalkyne is of any of FormulasII-XXII, above.

Second Reactant

The second reactant is a compound that comprises an azide such that acovalent bond is formed between the first and second reactants byreaction of the cycloalkyne moiety with the azide group. In general, thesecond reactant is of the formula:R₂—N₃

where R₂ is a target molecule, e.g., a biomolecule or other targetmolecule.

Target Molecules

Molecules comprising an azide and suitable for use in the presentinvention, as well as methods for producing azide-comprising moleculessuitable for use in the present invention, are well known in the art.Target molecules of particular interest as the second reactant include,but are not necessarily limited to, amino acids and amino acid residues,polypeptides (including peptides and proteins), sugars or sugarresidues, and the like, which contain or are modified to contain atleast one azide.

The target molecules can be naturally occurring, or may be syntheticallyor recombinantly produced, and may be isolated, substantially purified,or present within the native milieu of the unmodified molecule uponwhich the azide-containing target molecule is based (e.g., on a cellsurface or within a cell, including within a host animal, e.g., amammalian animal, such as a murine host (e.g., rat, mouse), hamster,canine, feline, bovine, swine, and the like). In some embodiments, thetarget molecule is present in vitro in a cell-free reaction. In otherembodiments, the target molecule is present in a cell and/or displayedon the surface of a cell. In many embodiments of interest, the targetmolecule is in a living cell; on the surface of a living cell; in aliving organism, e.g., in a living multicellular organism. Suitableliving cells include cells that are part of a living multicellularorganism; cells isolated from a multicellular organism; immortalizedcell lines; and the like.

Where the target molecule is a polypeptide, the polypeptide may becomposed of D-amino acids, L-amino acids, or both, and may be furthermodified, either naturally, synthetically, or recombinantly, to includeother moieties. For example, the target polypeptide may be alipoprotein, a glycoprotein, or other such modified protein.

In general, the target molecule useful as the second reactant comprisesat least one azide for reaction with modified cycloalkyne according tothe invention, but may comprise 2 or more, 3 or more, 5 or more, 10 ormore azides. The number of azides that may be present in a targetmolecule will vary according to the intended application of the finalproduct of the reaction, the nature of the target molecule itself, andother considerations which will be readily apparent to the ordinarilyskilled artisan in practicing the invention as disclosed herein.

This embodiment of the invention is particularly useful in modificationof a target molecule in vivo. In this embodiment, the target substrateis modified to comprise an azide group at the point at which linkage tothe modified cycloalkyne reactant is desired. For example, where thetarget substrate is a polypeptide, the polypeptide is modified tocontain an N-terminal azide. Where the target substrate is aglycoprotein, a sugar residue of the glycoprotein can be modified tocontain an azide. A target molecule that is unmodified with an azide,but that is to be modified with an azide, is referred to herein as a“target substrate.” A target molecule that is modified with an azide isreferred to herein as “an azide-modified target molecule” or “anazide-containing target molecule.”

Azide Modification of a Target Molecule

The target substrate can be generated in vitro and then introduced intothe cell using any of a variety of methods well known in the art (e.g.,microinjection, liposome or lipofectin-mediated delivery,electroporation, etc.), which methods will vary according to the natureof the substrate to be targeted for modification and can be readily andappropriately selected by the ordinarily skilled artisan. The finaltarget substrate can also be generated in vivo by exploiting a hostcell's natural biosynthetic machinery. For example, the cell can beprovided with a biocompatible azide-derivative of a substrate forsynthesis of the desired target molecule, which substrate is processedby the cell to provide an azide-derivative of the desired final targetsubstrate. For example, where the target substrate is a cell surfaceglycoprotein, the cell can be provided with an azide derivative of asugar residue found within the glycoprotein, which is subsequentlyprocessed by the cell through natural biosynthetic processes to producea modified glycoprotein having at least one modified sugar moietycomprising an accessible azide group.

The target substrate can also be produced in vivo using methods wellknown in the art. For example, unnatural amino acids having azides canbe incorporated into recombinant polypeptides expressed in E. coli (see,e.g., Kiick et al. (2000) Tetrahedron 56:9487). Such recombinantlyproduced polypeptides can be selectively reacted with a modifiedcycloalkyne reagent according to the invention.

In one example, an azide group is incorporated into the target moleculeby providing a cell (e.g., a eukaryotic cell that glycosylatesbiopolymers such as proteins) with a synthetic building block for thedesired biopolymer target substrate. For example, the cell can beprovided with a sugar molecule comprising an azide group to provide forincorporation of the azide group in a glycoprotein. In some embodiments,the glycoprotein is expressed on the cell surface. Alternatively, theazide group can be incorporated into an amino acid, which issubsequently incorporated into a peptide or polypeptide synthesized bythe cell. Several methods are available for incorporating unnaturalbuilding blocks into biopolymers; one need not be restricted to cellsurface oligosaccharides as target molecules. See, e.g., vanHest et al.(1998) FEBS Lett. 428:68; and Nowak et al. (1995) Science 268:439.

In one embodiment, the target molecule is a carbohydrate-containingmolecule (e.g., a glycoprotein; a polysaccharide; etc.), and an azidegroup is introduced into the target molecule using a syntheticsubstrate. In some embodiments, the synthetic substrate is an azidederivative of a sugar utilized in production of a glycosylated molecule.In some embodiments, the synthetic substrate is an azide derivative of asugar utilized in production of a cell surface molecule, e.g., in theglycoprotein biosynthetic pathway. For example, the host cell can beprovided with a synthetic sialic acid azido-derivative, which isincorporated into the pathway for sialic acid biosynthesis, eventuallyresulting in the incorporation of the synthetic sugar residue inglycoproteins. In some embodiments, the glycoproteins are displayed onthe cell surface.

In one example, the synthetic substrate is an azido derivative ofmannosamine of the general formula:

where n is from 1 to 6, generally from 1 to 4, more usually 1 to 2, andR₁, R₂, R₃, and R₄ are independently hydrogen or acetyl. In someembodiments, the substrate is N-azidoacetylmannosamine (n=1) or anacetylated derivative thereof, or N-azidopropanoylmannosamine (n=2) oran acetylated form thereof.

In another embodiment, the synthetic substrate is an azido sugarderivative of a general formula of, for example:

either of which can be incorporated into the sialic acid biosynthesispathway, and where n is from 1 to 6, generally from 1 to 4, more usually1 to 2, and R₂, R₃, and R₄ are independently hydrogen or acetyl.

In another embodiment, the synthetic substrate is an azido sugarderivative of a general formula of, for example:

where R₁, R₂, R₃, and R₄ are independently hydrogen or acetyl, and wherethe synthetic substrate is incorporated into biosynthetic pathwaysinvolving fucose.

In another embodiment, the synthetic substrate is an azido sugarderivative of a general formula of, for example:

where n is from 1 to 6, generally from 1 to 4, more usually 1 to 2, andR₁, R₂, R₃, and R₄ are independently hydrogen or acetyl, and which isincorporated into biosynthetic pathways involving galactose.

Cell Surface Modification

In some embodiments, a subject method is used to modify the surface of acell. Thus, in one aspect, the invention features a method of modifyingthe surface of cell in vitro or in vivo. The method generally involvesreacting an azide group in a target molecule that comprises an azidegroup with a modified cycloalkyne to provide for chemoselective ligationat the cell surface. In many embodiments, the method comprises modifyinga target molecule on a cell surface with an azide group; and reactingthe azide group in the target molecule with a modified cycloalkyne. Forexample, as described above, an azido sugar is provided to a livingcell, which azido sugar is incorporated into a glycoprotein that isdisplayed on the cell surface.

Modification of an Azide-Modified Target Molecule with DetectableLabels, Drugs, and Other Molecules

In some embodiments, the present invention provides for attachment of amolecule of interest, e.g., a functional molecule, to an azide-modifiedtarget molecule. The methods generally involve reacting anazide-modified target molecule with a subject modified cycloalkyne,where the modified cycloalkyne comprises a molecule of interest, asdescribed above. As described above, molecules of interest include, butare not limited to, a detectable label; a toxin (including cytotoxins);a linker; a peptide; a drug; a member of a specific binding pair; anepitope tag; and the like.

Attachment of Target Molecules to a Support

The modified cycloalkyne can also comprise one or more hydrocarbonlinkers (e.g., an alkyl group or derivative thereof such as an alkylester) conjugated to a moiety providing for attachment to a solidsubstratum (e.g., to facilitate assays), or to a moiety providing foreasy separation (e.g., a hapten recognized by an antibody bound to amagnetic bead). In one embodiment, the methods of the invention are usedto provide for attachment of a protein (or other molecule that containsor can be modified to contain an azide) to a chip in a definedorientation. For example, a polypeptide having an azide at a selectedsite (e.g., at or near the N-terminus) can be generated, and the methodsand compositions of the invention used to deliver a tag or other moietyto the azide of the polypeptide. The tag or other moiety can then beused as the attachment site for affixing the polypeptide to a support(e.g., solid or semi-solid support, particular a support suitable foruse as a microchip in high-throughput assays).

Attachment of Molecules for Delivery to a Target Site

The modified cycloalkyne will in some embodiments comprise a smallmolecule drug, toxin, or other molecule for delivery to a cell. Thesmall molecule drug, toxin, or other molecule will in some embodimentsprovide for a pharmacological activity. The small molecule drug, toxin,or other molecule will in some embodiments serve as a target fordelivery of other molecules.

Small molecule drugs may be small organic or inorganic compounds havinga molecular weight of more than 50 and less than about 2,500 daltons.Small molecule drugs may comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andmay include at least an amine, carbonyl, hydroxyl or carboxyl group, andmay contain at least two of the functional chemical groups. The drugsmay comprise cyclical carbon or heterocyclic structures and/or aromaticor polyaromatic structures substituted with one or more of the abovefunctional groups. Small molecule drugs are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

In another embodiment, a subject modified cycloalkyne comprises one of apair of binding partners (e.g., a ligand; a ligand-binding portion of areceptor; an antibody; an antigen-binding fragment of an antibody; anantigen; a hapten; a lectin; a lectin-binding carbohydrate; etc.). Forexample, the modified cycloalkyne can comprise a polypeptide that servesas a viral receptor and, upon binding with a viral envelope protein orviral capsid protein, facilitates attachment of virus to the cellsurface on which the modified cycloalkyne is displayed. Alternatively,the modified cycloalkyne comprises an antigen that is specifically boundby an antibody (e.g., monoclonal antibody), to facilitate detectionand/or separation of host cells displaying the antigen on the cellsurface. In another example, the modified cycloalkyne comprises a ligandbinding portion of a receptor, or a receptor-binding portion of aligand.

Utility

Subject modified cycloalkyne compounds, and subject modificationmethods, are useful in a variety of applications, including researchapplications and diagnostic applications.

Research Applications

In some embodiments, subject modified cycloalkyne compounds, and subjectmodification methods, are useful in research applications. Applicationsof interest include research applications, e.g., exploring functionaland physical characteristics of a receptor; proteomics; metabolomics;and the like. Research applications also include drug discovery or otherscreening applications.

Proteomics aims to detect, identify, and quantify proteins to obtainbiologically relevant information. Metabolomics is the detection,identification, and quantification of metabolites and other smallmolecules such as lipids and carbohydrates. Fiehn (2001) Comparative andFunctional Genomics 2:155-168; and U.S. Pat. No. 6,873,914.

Drug discovery applications include, but are not limited to, identifyingagents that inhibit cancer cell viability and/or growth. Thus, in someembodiments, the instant invention provides methods of identifying anagent that inhibits cancer cell viability and/or growth. The methodsgenerally involve modifying a component of the cell to comprise a firstreactive partner comprising an azide; contacting the cell, in thepresence of a test agent, with a second reactive partner comprising amodified cycloalkyne, the contacting being under physiologicalconditions; where the contacting results in reaction between the azidegroup of the first reactive partner and the cycloalkyne of the secondreactive partner, thereby synthetically and covalently modifying thecellular component; and determining the effect, if any, of the testagent on the level of modification of the cell with the second reactivepartner.

Where the cancer cell is one that produces a higher amount of acarbohydrate than a normal (non-cancerous) cell of the same cell type,the method provides for identifying an agent that reduces growth and/orviability of the cancerous cell.

Diagnostic and Therapeutic Applications

Applications of interest also include diagnostic applications, e.g., fordetection of cancer; and the like, where a subject modified cycloalkynecomprising a detectable label is used to label an azide-modified targetmolecule, e.g., an azide-labeled target molecule present on a cancercell. Applications of interest also include therapeutic applications,where a drug or other therapeutic agent is delivered to anazide-modified target molecule, using a subject modified cycloalkynethat comprises a covalently linked drug or other therapeutic agent.

In some embodiments, the present invention is used for in vivo imaging,e.g., to determine the metabolic or other state of a cell in anorganism, e.g., an individual. As one non-limiting example, a subjectmethod can be applied to in vivo imaging of cancer cells in anindividual (e.g., a mammal, including rodents, lagomorphs, felines,canines, equines, bovines, ovines, caprines, non-human primates, andhumans).

One exemplary, non-limiting application of a subject azide-alkynecycloaddition is in the detection of metabolic change in cells thatoccur as they alter their phenotype. As one example, alteredglycosylation patterns are a hallmark of the tumor phenotype, consistingof both the under- and over-expression of naturally-occurring glycans aswell as the presentation of glycans normally restricted to expressionduring embryonic development. Examples of common antigens associatedwith transformed cells are sialyl Lewis a, sialyl Lewis x, sialyl T,sialyl Tn, and polysialic acid (PSA). Jørgensen et al. (1995) CancerRes. 55, 1817-1819; Sell (1990) Hum. Pathology 21, 1003-1019; Taki etal. (1988) J. Biochem. 103, 998-1003; Gabius (1988) Angew. Chem. Int.Ed. Engl. 27, 1267-1276; Feizi (1991) Trends Biochem. Sci. 16, 84-86;Taylor-Papadimitriou and Epenetos (1994) Trends Biotech. 12, 227-233;Hakomori and Zhang (1997) Chem. Biol. 4, 97-104; Dohi et al. (1994)Cancer 73, 1552. These antigens share an important feature—they eachcontain terminal sialic acid. PSA is a homopolymer of sialic acidresidues up to 50 units in length. Elevated levels of sialic acid arehighly correlated with the transformed phenotype in many cancers,including gastric (Dohi et al. (1994) Cancer 73, 1552; and Yamashita etal. (1995) J. Natl. Cancer Inst. 87, 441-446), colon (Yamashita et al.(1995) J. Natl. Cancer Inst. 87, 441-446; Hanski et al. (1995) CancerRes. 55, 928-933; Hanski et al. (1993) Cancer Res. 53, 4082-4088; Yanget al. (1994) Glycobiology 4, 873-884; Saitoh et al. (1992) J. Biol.Chem. 267, 5700-5711), pancreatic (Sawada et al. (1994) Int. J. Cancer57, 901-907), liver (Sawada et al. (1994) J. Biol. Chem. 269,1425-1431), lung (Weibel et al. (1988) Cancer Res. 48, 4318-4323),prostate (Jørgensen et al. (1995) Cancer Res. 55, 1817-1819), kidney(Roth et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2999-3000), andbreast cancers (Cho et al. (1994) Cancer Res. 54, 6302-6305), as well asseveral types of leukemia (Joshi et al. (1987) Cancer Res. 47,3551-3557; Altevogt et al. (1983) Cancer Res. 43, 5138-5144; Okada etal. (1994) Cancer 73, 1811-1816). A strong correlation between the levelof cell surface sialic acid and metastatic potential has also beenobserved in several different tumor types (Kakeji et al. (1995) Brit. J.Cancer 71, 191-195; Takano et al. (1994) Glycobiology 4, 665-674). Thecollective display of multiple sialylated antigens on a single cancercell can account for the fact that so many different tumor types sharethe high sialic acid phenotype without necessarily expressing anidentical complement of antigens (Roth et al. (1988) supra).Consequently, diagnostic or therapeutic strategies that target cells onthe basis of sialic acid levels have broad applicability to manycancers.

Introduction and incorporation of unnatural azidosugars (ManNAz, GalNAz)into living animals provides for detection of changes in metabolicstate. Via the attachment of the appropriate epitope tag, the modifiedcyclooctyne labels these cells in a living organism, and consequentlydetects changes in metabolic state. Early detection of tumorigenic cellsand subsequent intervention reduces the severity and increases survivalrates for cancer patients.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Modification of Biomolecules Using a Modified CyclooctyneMaterials and Methods

All chemical reagents were purchased from Aldrich and used withoutfurther purification. All solvents were distilled under a N₂ atmosphere.CH₂Cl₂, toluene, and pyridine were dried over CaH₂. Thin layerchromatography was carried out on Analtech Uniplate® silica gel plates.Flash chromatography was performed using Merck 60 Å 230-400 mesh silicagel. All ¹H and ¹³C NMR spectra were acquired on Bruker AVB-400® orDRX-500® as noted. ¹H chemical shifts are reported as δ referenced tosolvent and coupling constants (J) are reported in Hz. Compounds 1 and 4have been previously reported. Skattebol and Solomon (1973) OrganicSyntheses 5/Coll. Volumes:306-310; and Wilbur et al. (1996) Bioconj.Chem. 7:689-702.

RPMI 1640 media and phosphate-buffered saline (PBS) were purchased fromInvitrogen Life Technologies. Fetal calf serum (FCS) was from Hyclone.FITC-conjugated avidin and bovine serum albumin (BSA) were purchasedfrom Sigma, and peroxidase-conjugated mouse anti-biotin (HRP-α-biotin)and peroxidase-conjugated donkey anti-human Ig (HRP-α-Ig) were fromJackson ImmunoResearch Laboratories, Inc. Precast Tris.HCl 4-15%polyacrylamide gels, nitrocellulose, and Tween 20 (non-ionic detergent)were purchased from BioRad. Restore™ Western blot stripping buffer andenhanced chemiluminescent substrate were obtained from Pierce. Forcell-labeling experiments, a Coulter Z2 cell counter was used todetermine cell densities. Flow cytometry data were acquired on a BDBiosciences FACSCalibur flow cytometer equipped with a 488-nm argonlaser, and live cells were analyzed as determined by granularity andsize. For all flow cytometry experiments, data points were collected intriplicate.

Synthesis of Compounds:

Compound 2. AgClO₄ (1.16 g, 5.61 mmol) was added to a solution ofcompound 1 (500 mg, 1.87 mmol) and methyl 4-hydroxymethylbenzoate (4.66g, 28.1 mmol) dissolved in toluene (8 mL) in a flame-dried,aluminum-foil-wrapped flask. The reaction was stirred for 2 h, dilutedwith pentane (20 mL), and filtered to remove insoluble silver salts. Thesolution was concentrated and purified by silica gel chromatography(4-8% EtOAc: pet ether; R_(f) (8% EtOAc: pet ether)=0.32) to yield 2 asa colorless oil (660 mg 1.86 mmol, 99%). ¹H NMR (500 MHz, CDCl₃) δ 8.02(d, 2H, J=8.0 Hz), 7.47 (d, 2H, J=8.0 Hz), 6.21 (dd, 1H, J=4.0, 11.5Hz), 4.72 (d, 1H, J=12.5 Hz), 4.39 (d, 1H, J=12.5 Hz), 3.95-3.91 (m,4H), 2.81 (app dq, 1H, J=5.5, 12.0), 2.32 (m, 1H), 2.05-1.88 (m, 4H),1.75 (m, 1H), 1.49 (app dq, 1H, J=5.0, 8.0), 1.35 (m, 1H), 0.79 (m, 1H);¹³C NMR (125 MHz, CDCl₃) δ 166.93, 143.20, 132.60, 132.09, 129.66,129.34, 127.65, 84.22, 69.61, 52.04, 39.48, 36.46, 33.31, 28.09, 26.29;IR (cm⁻¹) 2931, 2856, 1724, 1614, 1434, 1279, 1109; FAB HRMS calcd. forC₁₇H₂₃O₃Br [M+H]⁺ 353.0752. found 353.0744.

Compound 3. A suspension of NaOMe (119 mg, 2.20 mmol) in anhydrous DMSO(8 mL) was added to compound 2 (650 mg, 1.84 mmol) dissolved inanhydrous DMSO (16 mL). The reaction was stirred 20 min and additionalNaOMe (110 mg, 1.10 mmol in 1.5 mL of DMSO) was added. The reaction wasstirred until the starting material was completely consumed asdetermined by TLC (20 min). The reaction was acidified with 1 M HCl (100mL) and extracted twice with EtOAc (40 mL). The combined organic layerswere washed with brine (40 mL) and concentrated. The resulting film wasdissolved in 20% H₂O/dioxane (15 mL), LiOH (220 mg, 9.0 mmol) was added,and the reaction was stirred for 24 h. The reaction was acidified with 1M HCl (100 mL) and extracted twice with EtOAc (40 mL). The combinedorganic extracts were dried over MgSO₄, filtered, and purified by silicagel chromatography (10:90:1 EtOAc:pet ether: AcOH −25:75:1 EtOAc:petether: AcOH; R_(f) (25:75:1)=0.38) to yield 3 (350 mg, 1.36 mmol, 74%)as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.09 (d, 1H, J=8.50 Hz),7.47 (d, 1H, J=8.0 Hz), 4.77 (d, 1H, J=12.5 Hz), 4.49 (d, 1H, J=13 Hz),4.26 (m, 1H), 2.32-2.26 (m, 1H), 2.22-2.13 (m, 2H), 2.08-2.03 (m, 1H),1.97-1.92 (m, 1H), 1.89-1.83 (m, 2H), 1.73-1.62 (m, 2H) 1.52-1.44 (m,1H); ¹³C NMR (125 MHz, CDCl₃) δ 171.52, 144.50, 130.27, 128.29, 127.46,100.69, 92.40, 72.11, 70.39, 42.31, 34.26, 29.68, 26.31, 20.70; IR(cm⁻¹) 2924, 2852, 2672, 2561, 1685, 1429, 1323, 1294, 1086; FAB HRMScalcd. for C₁₆H₁₈O₃Li [M+Li]⁺ 265.1416. found 265.1422.

Compound 5. Pentafluorophenyl trifluoroacetate (Pfp-TFA) (40 μL, 0.23mmol) was added to compound 3 in anhydrous pyridine (1 mL). The reactionwas stirred for 4 h, diluted with CH₂Cl₂ (30 mL), and extracted with 1 MHCl (3×20 mL) and sat. NaHCO₃ solution (2×20 mL). The organic layer wasdried over MgSO₄, filtered, and concentrated. The crude ester wasdissolved in CH₂Cl₂ (2 mL). Triethylamine (67 μL, 0.46 mmol) andcompound 4 (104 mg 0.23 mmol) were added and the solution was stirredfor 1 h. The solution was concentrated and crude product purified bysilica gel chromatography (80:15:5 EtOAc:MeOH:H₂O, R_(f)=0.40) to yield5 (68 mg, 0.099 mmol, 51%) as a clear oil. ¹H NMR (400 MHz, CD₃CN) δ7.80 (d, 2H, J=8.4 Hz), 7.42 (m, 3H), 6.62 (br s, 1H), 5.51 (s, 1H),5.19 (s, 1H), 4.66 (d, 1H, J=12.0 Hz), 4.45-4.40 (m, 2H), 4.29-4.26 (m,2H), 3.62-3.56 (m, 8H), 3.52-3.49 (m, 2H), 3.47-3.42 (m, 4H), 3.22-3.13(m, 3H), 2.91-2.87 (m, 2H), 2.66 (d, 1H, J=12.8 Hz), 2.32-2.19 (m, 6H),1.89-1.79 (m, 4H), 1.75-1.34 (m, 10H); ¹³C NMR (125 MHz, CD₃CN) δ172.64, 166.56, 162.93, 141.82, 134.15, 127.47, 127.05, 100.31, 92.63,71.94, 70.12, 69.94, 69.83, 69.16, 68.75, 61.37, 59.80, 55.36, 42.14,40.18, 37.53, 36.56, 35.45, 34.12, 29.56, 29.32, 29.26, 28.02, 27.98,26.18, 25.46, 20.13; FAB HRMS calcd. for C₃₆H₅₅N₄O₇S [M+H]⁺ 687.3791.found 687.3798.

Compound 6. Compound 4 (46 mg, 0.10 mmol), pentynoic acid (15 mg, 0.15mmol) and HATU (59 mg, 0.15 mmol) were dissolved in anhydrous DMF (1mL). Triethyl amine (22 μL, 0.15 mmol) was added to the solution and thereaction was stirred for 14 h. Purification by silica gel chromatography(15:2:1 EtOAc:MeOH:H₂O to 9:2:1 EtOAc:MeOH:H₂O R_(f) (9:2:1)=0.30)yielded 6 (25 mg, 0.047 mmol, 46%) as a yellow oil. ¹H NMR (400 MHz,CD₃CN) δ 6.77 (br s, 2H), 5.74 (s, 1H), 5.38 (s, 1H), 4.44 (app t, 1H,J=7.6 Hz), 4.27 (m, 1H), 3.61-3.54 (m, 8H), 3.51 (t, 4H, J=6.4 Hz),3.25-3.16 (m, 5H), 2.91 (dd, 1H, J=4.8, 12.4 Hz), 2.68 (d, 1H, J=12.8Hz), 2.47 (m, 2H), 2.35-2.30 (m, 6H), 2.22 (t, 1H, J=2.8 Hz), 2.17 (t,2H, J=7.2 Hz), 1.75-1.53 (m, 8H), 1.44-1.37 (m, 2H); ¹³C NMR (100 MHz,CD₃CN) δ 172.79, 170.74, 163.21, 83.53, 70.10, 69.82, 69.10, 68.71,68.60, 61.46, 59.87, 55.41, 40.18, 36.49, 35.47, 34.69, 29.32, 29.29,28.07, 28.01, 25.48, 14.32; FAB LRMS calcd. for C₂₅H₄₃N₄O₆S [M+H]⁺527.4. found 527.4.

Western Blot Analysis of an Azide-Labeled GlyCAM-Ig

For Western blot analysis of azide-labeled and unlabeled GlyCAM-Ig,samples were incubated with 5 (250 μM final concentration in PBS, pH 7.4containing 0.7% DMF), 6 (250 μM final concentration in PBS, pH 7.4containing either 0.7% DMF) or left untreated for 12 h. To verify that 6could react with azide-labeled GlyCAM-Ig via copper-mediated [3+2]cycloaddition, a modified version of the method reported by Speers andCravatt was employed. Speers and Cravatt (2004) Chem. Biol. 11:535-46.Briefly, GlyCAM-Ig samples (in PBS, pH 7.4 containing 0.7% DMF) wereincubated with 250 μM 6, 2.5 mM TCEP, 250 μM tris-triazolyl ligand (froma 1.7 mM stock in 1:4 DMSO:tert-butanol), and 2.5 mM CuSO₄. Samples werevortexed and allowed to react at rt for 12 h. Prior to electrophoresis,samples were incubated with an equal volume of 100 mM 2-azidoethanol in2×SDS-PAGE loading buffer for 8 h at rt (to quench unreacted 5 or 6).The quenched samples were boiled for 3 min and loaded onto pre-castpolyacrylamide gels. After electrophoresis, the samples wereelectroblotted to nitrocellulose membrane. The membrane was blockedusing 5% BSA in PBS (pH 7.4, containing 0.05% Tween 20 (blocking bufferA) for 1 h at rt, then incubated with a solution of blocking buffer Acontaining HRP-α-biotin (1:250,000 dilution, 1 h at rt). The membranewas washed, and detection of membrane-bound anti-biotin-HRP wasaccomplished by chemiluminescence. Following detection, the membrane wasrinsed with PBS (pH 7.4) containing 0.05% Tween (PBS-T) and placed instripping buffer (15 min at RT) to remove bound anti-biotin Ig. Themembrane was thoroughly rinsed with PBS-T and probed for residualanti-biotin signal by chemiluminescence.

The membrane was washed and re-blocked with 5% low-fat dry powdered milk(blocking buffer B) for 1 h at rt. The membrane was then incubated withHRP-α-Ig (1:5000 in blocking buffer B) for 1 h at rt. The membrane waswashed again with PBS-T, and detection of membrane bound anti-human Igwas accomplished as for peroxidase-conjugated anti-biotin. Controlsamples were treated in an identical manner, except that specificreagents were replaced by buffer where appropriate.

Cell Culture Conditions

Jurkat cells (human T-cell lymphoma) were maintained in a 5% CO₂,water-saturated atmosphere and grown in RPMI-1640 media supplementedwith 10% FCS, penicillin (100 units/mL) and streptomycin (0.1 mg/mL).Cell densities were maintained between 1×10⁵ and 1.6×10⁶ cells/mL forall experiments.

Cell Surface Azide Labeling and Detection

Jurkat cells were seeded at a density of 1.5-2.0×10⁵ cells/mL andincubated for 3 d in untreated media or media containing variousconcentrations of Ac₄ManNAz. After growth in the presence of Ac₄ManNAz,cells were distributed into a 96-well V-bottom tissue culture plate. Thecells were pelleted (3500 rpm, 3 min) and washed twice with 200 μL oflabeling buffer (PBS, pH 7.4 containing 1% FCS). Cells were thenincubated with 5 or a biotinylated phosphine probe (Vocadlo et al.(2003) Proc. Natl. Acad. Sci. U.S.A. 100:9116-21) in labeling buffercontaining 2.8% DMF or labeling buffer plus 2.8% DMF alone. Afterincubation, cells were pelleted, washed twice with labeling buffer andresuspended in the same buffer containing FITC-avidin (1:500 dilution ofthe Sigma stock). Following a 10-min incubation on ice (in the dark),cells were washed with 200 μL of cold labeling buffer and theFITC-avidin staining procedure was repeated. The cells were washed twicewith cold labeling buffer, then diluted to a volume of 400 μL for flowcytometry analysis.

Results

Biotinylated cyclooctyne 5 as synthesized as shown in Scheme 1.

Construction of the substituted cyclooctyne core was achievedessentially as described

by Reese and Shaw. Reese and Shaw (1970) Chem. Comm. 1172-1173. Briefly,compound 1 (Skattebol and Solomon, supra) was treated with silverperchlorate to effect electrocyclic ring opening to the trans-allyliccation, which was captured with methyl hydroxymethylbenzoate to affordbromo-trans-cyclooctene 2. Base-mediated elimination of the vinylbromide followed by saponification yielded versatile intermediate 3, towhich any biological probe can be attached. Finally, compound 3 wascoupled to biotin analog 4 (Wilbur et al. (1996) Bioconj. Chem.7:689-702) bearing a PEG linker, providing target 5. Cyclooctyne 3 wasstable to mild acid (0.5 N HCl for 30 min), base (0.8 M NaOMe for 30min), and prolonged exposure to biological nucleophiles such as thiols(120 mM 2-mercaptoethanol for 12 h).

Model reactions were performed with compound 3 and 2-azidoethanol,benzylazide or N-butyl α-azidoacetamide. In all cases, the only productsobserved were the two regioisomeric triazoles formed in approximatelyequal amounts. The reaction was then applied for covalent labeling ofbiomolecules. The recombinant glycoprotein GlyCAM-Ig (Bistrup et al.(1999) J. Cell Biol. 145:899-910) was expressed in CHO cells in thepresence of peracetylated N-azidoacetylmannosamine (Ac₄ManNAz), leadingto metabolic incorporation of the corresponding N-azidoacetyl sialicacid (SiaNAz) into its glycans. Control samples of GlyCAM-Ig wereexpressed in the absence of azido sugar. The purified GlyCAM-Ig sampleswere incubated with 250 μM 5 overnight, the unreacted cyclooctyne wasquenched with excess 2-azidoethanol, and the samples were analyzed byWestern blot probing with HRP-α-biotin (FIG. 2). Robust biotinylationwas observed for GlyCAM-Ig modified with SiaNAz. Native GlyCAM-Iglacking azides showed no background labeling, underscoring the exquisiteselectivity of the strain-promoted cycloaddition.

As a point of comparison, similar reactions were performed withbiotin-modified terminal alkyne 6 (Scheme 1). In the absence of reagentsfor copper catalysis, no glycoprotein labeling was observed (FIG. 2). Asexpected based on previous reports (Sharpless and Finn (2003) J. Am.Chem. Soc. 125:3192-3193; and Speers and Cravatt ((2004) Chem. Biol.11:535-546), addition of CuSO₄, TCEP and a triazolyl ligand resulted infacile labeling of the azide-modified glycoprotein. The blot wasstripped and reprobed with HRP-labeled anti-IgG antibody (HRP-α-IgG) toconfirm equal protein loading. Interestingly, consistently diminishedanti-IgG immunoreactivity was observed for azide-modified GlyCAM-Iglabeled with 6 in the presence of copper. It is possible that thecombination of triazole products and copper damages the epitoperecognized by HRP-α-IgG.

Finally, the utility of the strain-promoted reaction for live celllabeling was investigated. Jurkat cells were incubated with 25 μMAc₄ManNAz for 3 d in order to introduce SiaNAz residues into their cellsurface glycoproteins. Saxon and Bertozzi (2000) Science 287:2007-2010;and Saxon et al. 92002) J. Am. Chem. Soc. 124:14893-14902. The cellswere reacted with various concentrations of 5 for 1.5 h at rt, thenstained with FITC-avidin and analyzed by flow cytometry. As shown inFIG. 3A, cells displaying azides showed a dose-dependent increase influorescence upon treatment with the cyclooctyne probe. No detectablelabeling of cells lacking azides was observed. The cell surface reactionwas also dependent on duration of incubation with 5 (FIG. 3B) and thedensity of cell-surface azides. No negative effects on cell viabilitywere observed.

FIG. 2. Labeling of azide-modified GlyCAM-Ig with alkyne probes.Purified GlyCAM-Ig was treated with buffer (−), 250 μM 5, or 250 μM 6alone or in the presence (cat) of CuSO₄, TCEP, and a triazolyl ligand,overnight at rt. Reaction mixtures were quenched with 2-azidoethanol andanalyzed by Western blot probing with HRP-α-biotin (upper panel). Theblot was then stripped and reprobed with HRP-α-Ig (lower panel).

FIGS. 3A and 3B. Cell surface labeling with compound 5. Jurkat cellswere incubated in the presence (+Az) or absence (−Az) of 25 μM Ac₄ManNAzfor 3 d. FIG. 3A. The cells were reacted with various concentrations of5 for 1 h at rt and treated with FITC-avidin; mean fluorescenceintensity (MFI) was determined by flow cytometry. FIG. 3B. The cellswere incubated with 250 μM 5 for various durations at rt and analyzed asin A. C. The cells were incubated with 100 μM probe for 1 h at rt andanalyzed as in A. Error bars represent the standard deviation from threereplicates. AU=arbitrary fluorescence units.

The above example demonstrates that [3+2] cycloaddition of azides andcyclooctyne derivatives (modified cyclooctynes) according to a subjectmethod occurs readily under physiological conditions in the absence ofauxiliary reagents; and that the selective chemical modification ofliving cells using a subject method occurs without any apparenttoxicity.

Example 2 Synthesis of Additional Cyclooctyne Compounds and their Use inLabeling of Living Cells

This example presents the synthesis of compounds 3a and 3b (shownbelow), the evaluation of their kinetic parameters in reactions withsmall organic azides, and their use in bioorthogonal labeling of livingcells.

Materials and Methods

All chemical reagents were of analytical grade, obtained from commercialsuppliers, and used without further purification unless otherwise noted.With the exception of reactions performed in aqueous media, all reactionvessels were flame-dried prior to use. Reactions were performed in a N₂atmosphere, except in the case of reactions performed in aqueous media,and liquid reagents were added with a syringe unless otherwise noted.Tetrahydrofuran (THF) was distilled under N₂ from Na/benzophenoneimmediately prior to use, and CH₂Cl₂ was distilled from CaH₂ immediatelyprior to use. Chromatography was carried out with Merck 60 230-400 meshsilica gel according to the procedure described by Still. J. Org. Chem.(1978) 43:2923-2925. Reactions and chromatography fractions wereanalyzed with Analtech 250 micron silica gel G plates, and visualized bystaining with ceric ammonium molybdate or by absorbance of UV light at245 nm. When an R_(f) is reported for a compound, the solvent that wasused was the chromatography solvent unless otherwise noted. Organicextracts were dried over MgSO₄ and solvents were removed with a rotaryevaporator at reduced pressure (20 torr), unless otherwise noted. IRspectra were of thin films on NaCl plates. Unless otherwise noted, ¹H,¹³C, and ¹⁹F NMR spectra were obtained with 300 MHz or 400 MHz Brukerspectrometers. Chemical shifts are reported in δ ppm referenced to thesolvent peak for ¹H and ¹³C and relative to CFCl₃ for ¹⁹F. Couplingconstants (J) are reported in Hz. Low- and high-resolution fast atombombardment (FAB) and electron impact (EI) mass spectra were obtained atthe UC Berkeley Mass Spectrometry Facility, and elemental analyses wereobtained at the UC Berkeley Micro-Mass Facility. Melting points weredetermined using a MeI-Temp 3.0 melting point apparatus and areuncorrected.

Synthetic Procedures

(Cyclooct-1-enyloxy)-trimethylsilane (6). To a solution of cyclooctanone(40.0 g, 316 mmol) in 200 mL of anhydrous DMF were added triethylamine(TEA, 93.0 mL, 666 mmol) and chlorotrimethylsilane (84.0 mL, 666 mmol).The reaction was heated to reflux. After 15 h, the reaction was quenchedwith 20 mL of H₂O and the DMF was removed on a rotary evaporator. Theresidue was diluted with hexanes (300 mL), washed with H₂O (3×100 mL)and brine (1×50 mL), and dried over MgSO₄. Distillation under reducedpressure (20 torr) yielded 58.1 g (93%) of the desired product as acolorless oil, bp 108° C. (20 torr) (lit. 106° C. at 25 torr). Nakamura,et al. J. Am. Chem. Soc. (1976) 98, 2346-2348. IR: 2926, 2851, 1661cm⁻¹. ¹H NMR (CDCl₃, 300 MHz): δ 0.20 (s, 9H), 1.39-1.58 (m, 8H), 2.02(m, 2H), 2.19 (m, 2H), 4.75 (t, 1H, J=9.0 Hz). (Lit: ¹H NMR (CDCl₃, 600MHz): δ 0.16 (s, 9H), 1.46 (m, 4H), 1.49 (m, 2H), 1.55 (m, 2H), 1.97 (m,2H), 2.14 (m, 2H), 4.70 (t, 1H, J=8.0 Hz) (Frimer et al. J. Org. Chem.(2000) 65, 1807-1817.) ¹³C NMR (CDCl₃, 75 MHz): δ 0.4, 25.5, 26.3, 26.4,27.8, 30.9, 31.0, 105.41, 152.99. (Lit: ¹³C NMR (CDCl₃, 150 MHz): δ0.45, 25.52, 26.36, 26.40, 27.83, 30.98, 31.05, 105.45, 153.05) Frimeret al. (2000) supra) FAB-HRMS: Calcd. for C₁₁H₂₃OSi⁺ [M+H]⁺: 199.1518.found 199.1520.

2-Fluorocyclooctanone (5b). To a solution of silyl enol ether 6 (57.8 g,291 mmol) in DMF (350 mL) was added a solution of Selectfluor (124 g,349 mmol) in DMF (150 mL) over 1 h at rt. The solution was allowed tostir for 12 h. The reaction was quenched with 30 mL of H₂O, and the DMFwas removed on a rotary evaporator. The residue was diluted with hexanes(500 mL), washed with H₂O (3×200 mL) and brine (1×50 mL), and dried overMgSO₄. Following column chromatography (30:1 to 15:1 pentane/ether), awhite solid was isolated (38.4 g, 91%, R_(f)=0.30 in 9:1 pentane/ether),mp 48.1-49.8° C. IR: 3429, 2932, 2859, 1721, 1709 cm¹. (Lit: 1710 cm⁻¹)(Rozen and Menahem. J. Fluorine Chem. (1980), 16, 19-31). ¹H NMR (CDCl₃,400 MHz): δ 1.25 (m, 1H), 1.38-1.55 (m, 4H), 1.61 (m, 1H), 1.73 (m, 1H),2.05 (m, 3H), 2.30 (m, 1H), 2.55 (m, 1H), 4.86 (ddd, 1H, J=49.6, 6.4,2.8 Hz). (Lit: ¹H NMR (CDCl₃, 250 MHz): δ 1.36-2.7 (m, 12H), 4.9 (dm,1H, J=49.5 Hz) (Chambers and Hutchinson J. Fluorine Chem. (1998) 89,229-232) ¹³C NMR (CDCl₃, 100 MHz): δ 20.3 (d, J=4.0 Hz), 24.4 (d, J=6.0Hz), 24.5, 26.9, 32.2 (d, J=11.0 Hz), 39.3, 94.7 (d, J=185.0 Hz), 213.4(d, J=21.0 Hz). (Lit: ¹³C NMR (CDCl₃, 50 MHz): 620.5 (d, J=3.6 Hz), 24.6(d, J=3.7 Hz), 24.7, 27.2, 32.7 (d, J=21.0 Hz), 39.6, 91.5 (d, J=184.7Hz), 213.9 (d, J=20.9 Hz) (Chambers and Hutchinson (1998) supra) ¹⁹F NMR(CDCl₃, 376 MHz): δ −190.7 (m). (Lit: ¹⁹F NMR (CDCl₃, 235 MHz): δ −191.6(m) (Chambers and Hutchinson (1998) supra)

4-(1-Fluoro-2-oxo-cyclooctylmethyl)benzoic acid methyl ester (4b). To asolution of LDA (34 mL, 61 mmol, 1.8 M solution inheptane/THF/ethylbenzene) in THF (50 mL) at −78° C. was added a solutionof 2-fluorocyclooctanone (5b) (7.38 g, 51.2 mmol) in THF (50 mL) over 2h using a syringe pump. After 1 h, a solution of methyl4-bromomethylbenzoate (17.6 g, 76.8 mmol) in THF (50 mL) was added andthe reaction was allowed to warm to rt. After 1 h, the reaction mixturewas quenched with H₂O (50 mL) and the THF was removed on a rotaryevaporator. The residue was diluted with EtOAc (200 mL), washed with H₂O(3×100 mL) and brine (1×50 mL), and dried over MgSO₄. Following columnchromatography (50:1 to 15:1 hexanes/EtOAc), a white solid was isolated(6.45 g, 43%, R_(f)=0.35 in 9:1 hexanes/EtOAc), mp 53.5-55.2° C. IR:2931, 2858, 1721 cm⁻. ¹H NMR (CDCl₃, 400 MHz): δ 1.08 (m, 1H), 1.35-1.65(m, 7H), 1.86 (m, 2H), 2.04 (app d, 2H, J=9.0 Hz), 2.90 (dd, 1H, J=20.1,14.1 Hz), 3.14 (dd, 1H, J=28.2, 14.1 Hz), 3.78 (s, 3H), 7.15 (d, 2H,J=7.8 Hz), 7.83 (d, 2H, J=8.4 Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 21.1,24.7, 25.3, 27.6, 38.7 (d, J=22.0 Hz), 40.0, 43.4 (d, J=21.0 Hz), 51.9,102.3 (d, J=189.0 Hz), 128.7, 129.3, 130.5, 140.3, 166.8, 216.0 (d,J=26.0 Hz). ¹⁹F NMR (CDCl₃, 376 MHz): δ −166.0 (m). FAB-HRMS: Calcd. forC₁₇H₂₂O₃F+[M+H]⁺: 293.1553. found: 293.1560.

4-(1-Fluoro-2-trifluoromethanesulfonyloxy-cyclooct-2-enylmethyl)-benzoicacid methyl ester (7b). To a solution of ketone 4b (4.62 g, 15.8 mmol)in THF (50 mL) at −78° C. was added Potassium hexamethyldisilazide(KHMDS, 35.0 mL, 17.5 mmol, 0.50 M solution in toluene). After 1 h, asolution of N-Phenylbistrifluoromethanesulfonimide (Tf₂NPh, 6.18 g, 17.3mmol) in THF (25 mL) was added and the reaction was allowed to warm tort. After 30 min, the reaction mixture was concentrated on a rotaryevaporator and the product was purified by column chromatography (20:1to 12:1 hexanes/EtOAc) to yield the desired product (4.90 g, 73%,R_(f)=0.30 in 9:1 hexanes/EtOAc) as a colorless oil. IR: 3424, 2952,1723, 1646 cm⁻¹. ¹H NMR (CDCl₃, 400 MHz): δ 1.38-1.67 (m, 4H), 1.74-1.97(m, 4H), 1.97-2.18 (m, 2H), 3.16 (app d, 1H, J=3.2 Hz), 3.20 (s, 1H),3.91 (s, 3H), 5.86 (t, 1H, J=9.6 Hz), 7.26 (d, 2H, J=8.0 Hz), 7.96 (d,2H, J=8.4 Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 22.0, 22.1, 23.1, 25.2, 35.1(d, J=22.0 Hz), 44.3 (d, J=26.0 Hz), 52.0, 95.7 (d, J=177.0 Hz), 118.4(q, J=317.0 Hz), 123.8, 129.1, 129.4, 130.5, 139.7 (d, J=8.0 Hz), 148.2(d, J=21.0 Hz), 166.8. ¹⁹F NMR (CDCl₃, 376 MHz): δ −73.7 (app d, 3H,J=3.8 Hz), −144.3 (app d, 1H, J=3.8 Hz). FAB-HRMS: Calcd. forC₁₈H₂₁O₅F₄S⁺ [M+H]⁺: 425.1046. found: 425.1046.

4-(1-Fluoro-cyclooct-2-ynylmethyl)benzoic acid methyl ester (8b). To asolution of vinyl triflate 7b (14.3 g, 33.5 mmol) in THF (100 mL) at 0°C. was added LDA (23.5 mL, 35.2 mmol, 1.8M in heptane/THF/ethyl benzene)dropwise, using a syringe pump, over 3 h. LDA was added until thestarting material was consumed. The reaction was quenched with 20 mL ofH₂O and the THF was removed on a rotary evaporator. The residue wasdiluted with EtOAc (300 mL), washed with H₂O (3×100 mL) and brine (1×50mL), and dried over MgSO₄. After silica gel chromatography (50:1 to 25:1hexanes/EtOAc), a colorless oil was isolated (5.41 g, 59%, R_(f)=0.40 in9:1 hexanes/EtOAc). IR: 3421, 2930, 2854, 2223, 1721, 1613 cm⁻¹. ¹H NMR(CDCl₃, 300 MHz): δ 1.35 (m, 1H), 1.66-1.76 (m, 2H), 1.76-2.07 (m, 4H),2.07-2.33 (m, 3H), 3.03 (app d, 1H, J=2.1 Hz), 3.10 (s, 1H), 3.90 (s,3H), 7.37 (d, 2H, J=8.1 Hz), 7.98 (d, 2H, J=8.1 Hz). ¹³C NMR (CDCl₃, 75MHz): δ 20.5, 26.0 (d, J=1.5 Hz), 29.4, 34.0, 44.7 (d, J=26.0 Hz), 48.8(d, J=25.0 Hz), 51.9, 90.5 (d, J=32.0 Hz), 95.2 (d, J=175.0 Hz), 104.5(d, J=11.0 Hz), 128.6, 129.3, 130.3, 141.2, 167.0. ¹⁹F NMR (CDCl₃, 376MHz): δ −139.0 (m). FAB-HRMS: Calcd. for C₁₇H₂₀O₂F+[M+H]⁺: 275.1447.found: 275.1442.

4-(1-Fluoro-cyclooct-2-ynylmethyl)benzoic acid (3b). To a solution ofcyclooctyne 8b (1.8 g, 6.6 mmol) in dioxane (30 mL) and H₂O (7.5 mL) wasadded finely crushed LiOH (3.1 g, 130 mmol). The suspension was heatedto 50° C. and stirred for 3 h. The dioxane was removed on a rotaryevaporator and the reaction mixture was diluted with CH₂Cl₂ (100 mL).The organic layer was washed with 1 N HCl (2×100 mL), H₂O (3×100 mL),and brine (1×25 mL), and dried over MgSO₄, yielding a white solid (1.7g, 98%, R_(f)=0.30 in 27:3:1 hexane/EtOAc/AcOH), mp 132.0-132.5° C.(dec). IR: 3442, 2926, 2851, 2674, 2557, 2224, 1686, 1611 cm⁻¹. ¹H NMR(DMSO-d₆, 400 MHz): δ 1.39 (app quintet, 1H, J=7.6 Hz), 1.64 (m, 2H),1.76 (m, 2H), 2.10 (m, 5H), 3.07 (s, 1H), 3.11 (d, 1H, J=8.0 Hz), 7.38(d, 2H, J=8.0 Hz), 7.87 (d, 2H, J=8.4 Hz), 12.9 (br s, 1H). ¹³C NMR(DMSO-d₆, 100 MHz): δ 19.8, 25.7, 29.0, 33.7, 43.7 (d, J=25.0 Hz), 47.6(d, J=25.0 Hz), 90.7 (d, J=32.0 Hz), 95.4 (d, J=173.0 Hz), 104.5 (d,J=10.0 Hz), 129.0, 129.2, 130.4, 141.2, 167.2. ¹⁹F NMR (CD₃CN, 376 MHz):δ −139.4 (m). FAB-HRMS: Calcd. for C₁₆H₁₈O₂F+[M+H]⁺: 261.1291. found:261.1291. Anal. calcd. for C₁₆H₁₇O₂F: C, 79.31; H, 7.49. Found: C,79.07; H, 7.26.

3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propylammonium trifluoroacetate(13). To a solution of maleimide (3.12 g, 32.2 mmol) and PPh₃ (8.29 g,31.6 mmol) in THF (150 mL) was added N-(t-butoxycarbonyl)propanolamine(5.00 mL, 29.3 mmol) and then diisopropyl azodicarboxylate (6.80 mL,35.1 mmol). The solution was stirred at rt for 48 h, concentrated on arotary evaporator, and purified by column chromatography (4:1 to 2:1hexanes/EtOAc) to yield 10.3 g of a mixture of the N-Boc protectedproduct and diisopropyl hydrazinedicarboxylate. The mixture wasdissolved in a solution of CH₂Cl₂ (60 mL) and H₂O (5 mL), and thentrifluoroacetic acid (TFA, 35 mL) was added. The reaction was stirredfor 5 h at rt and then diluted with CH₂Cl₂ (50 mL) and H₂O (50 mL). Theaqueous layer was washed with CH₂Cl₂ (3×50 mL) and then concentrated toyield the desired product as a yellow oil (7.51 g, 96%). IR: 3435, 2959,2917, 2849, 1707, 1683 cm⁻¹. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.76 (app q,2H, J=7.2 Hz), 2.78 (m, 2H), 3.45 (t, 2H, J=6.8 Hz), 7.02 (s, 2H), 7.79(br s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz): δ 26.7, 34.6, 37.0, 116.0 (q,289.0 Hz), 134.7, 158.9 (q, J=36.0 Hz), 171.3. ¹⁹F NMR (DMSO-d₆, 376MHz): δ −72.9 (s). FAB-HRMS: Calcd. for C₇H₁₁N₂O₂ ⁺ [M+H]⁺: 155.0821.found: 155.0824.

N-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propyl]-4-(1-fluorocyclooct-2-ynylmethyl)benzamide(12b). TEA (0.508 mL, 3.65 mmol) was added to a solution of cyclooctyne3b (0.200 g, 0.768 mmol), amine 13 (0.235 g, 0.875 mmol),O-(7-Azabenzotriazol-1-yl)-N,N,N′,N-tetramethyluroniumhexafluorophosphate (HATU, 0.305 g, 0.802 mmol), and1-hydroxybenzotriazole (HOBT, 0.123 g, 0.802 mmol) in CH₂Cl₂ (3 mL) atrt. The reaction was stirred for 15 min at rt, quenched with H₂O (10mL), and diluted with CH₂Cl₂ (50 mL). The organic layer was washed with1 N HCl (3×50 mL), saturated NaHCO₃ (3×50 mL), and brine (2×25 mL) anddried over MgSO₄. Chromatography of the crude product (2:1 to 1:1hexanes/EtOAc) yielded the desired product as a white solid (0.198 g,65%, R_(f)=0.30 in 1:1 hexanes/EtOAc), mp 122.0-124.0° C. (dec). IR:3413, 2931, 2854, 2222, 1705, 1640 cm⁻¹. ¹H NMR (CDCl₃, 400 MHz): δ 1.39(m, 1H), 1.74 (m, 2H), 1.81-2.06 (m, 6H), 2.14-2.32 (m, 3H), 3.05 (d,1H, J=2.8 Hz), 3.09 (s, 1H), 3.40 (q, 2H, J=6.4 Hz), 3.67 (t, 2H, J=6.0Hz), 6.75 (s, 2H), 6.93 (m, 1H), 7.41 (d, 2H, J=8.0 Hz), 7.81 (d, 2H,J=8.0 Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 20.5, 26.0, 28.1, 29.4, 34.0,34.8, 36.2, 44.6 (d, J=25.0 Hz), 47.8 (d, J=24.0 Hz), 90.7 (d, J=32.0Hz), 95.3 (d, J=174.0 Hz), 104.5 (d, J=10.0 Hz), 126.7, 130.5, 132.9,134.2, 139.6, 167.1, 171.1. ¹⁹F NMR (CDCl₃, 376 MHz): δ −139.0 (m).FAB-HRMS: Calcd. for C₂₃H₂₆N₂O₃F+[M+H]⁺: 397.1927. found: 397.1920.

4-(2-Oxo-cyclooctylmethyl)benzoic acid methyl ester (4a). To a stirringsolution of cyclooctanone (1.76 g, 14.0 mmol) in THF (30 mL) at −78° C.was added LDA (8.54 mL, 15.4 mmol, 1.8 M in heptane/THF/ethylbenzene).After 1 h, a solution of methyl 4-bromomethylbenzoate (3.53 g, 15.4mmol) in THF (10 mL) was added and the reaction mixture was allowed towarm to rt. After 30 min, the reaction was quenched with H₂O (10 mL) andthe THF was removed on a rotary evaporator. The residue was diluted withEtOAc (100 mL), washed with H₂O (3×100 mL) and brine (1×50 mL), anddried over MgSO₄. Chromatography of the crude product (50:1 to 9:1hexanes/EtOAc) yielded the desired product as a colorless oil (3.08 g,80%, R_(f)=0.30, 9:1 hexanes/EtOAc). IR: 3426, 2929, 2856, 1721, 1700,1657, 1611 cm⁻¹. ¹H NMR (CDCl₃, 300 MHz): δ 1.10-1.28 (m, 1H), 1.28-1.42(m, 1H), 1.42-1.80 (m, 6H), 1.80-1.90 (m, 1H), 1.90-2.08 (m, 1H),2.08-2.18 (m, 1H), 2.22-2.35 (m, 1H), 2.64 (dd, 1H, J=12.6, 6.0 Hz),3.01 (m, 2H), 3.89 (s, 3H), 7.20 (d, 2H, J=8.1 Hz), 7.93 (d, 2H, J=8.4Hz). ¹³C NMR (CDCl₃, 75 MHz): δ 24.5, 24.6, 25.1, 27.7, 32.9, 38.1,43.2, 51.5, 52.0, 128.1, 129.0, 129.7, 145.6, 167.0, 218.9. FAB-HRMS:Calcd. for C₁₇H₂₃O₃ ⁺ [M+H]⁺: 275.1647. found: 275.1648.

4-(2-Trifluoromethanesulfonyloxy-cyclooct-2-enylmethyl)-benzoic acidmethyl ester (7a). To a solution of ketone 4a (2.17 g, 7.91 mmol) in THF(50 mL) at −78° C. was added KHMDS (17.4 mL, 8.70 mmol, 0.50 M solutionin toluene). After 1 h, a solution of Tf₂NPh (3.11 g, 8.70 mmol) in THF(25 mL) was added and the reaction was allowed to warm to rt. After 30min, the reaction mixture was concentrated on a rotary evaporator andthe product was purified directly by column chromatography (20:1 to 12:1hexanes/EtOAc) to yield the desired product (2.19 g, 68%, R_(f)=0.35 in9:1 hexanes/EtOAc) as a colorless oil. The product was isolated as a 9:1mixture of regioisomers. IR: 2932, 2856, 1723, 1410 cm⁻¹. ¹H NMR (CDCl₃,400 MHz): δ 1.25-1.38 (m, 1H), 1.38-1.52 (m, 1H), 1.52-1.71 (m, 4H),1.71-1.85 (m, 2H), 1.98-2.11 (m, 1H), 2.15-2.25 (m, 1H), 2.73 (dd, 1H,J=14.0, 6.8 Hz), 2.99 (dd, 1H, J=13.6, 8.0 Hz), 3.13 (m, 1H), 3.73 (s,3H), 5.76 (t, 1H, J=8.8 Hz), 7.27 (d, 2H, J=8.0 Hz), 7.98 (d, 2H, J=8.0Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 25.2, 25.9, 26.5, 29.8, 33.3, 37.4,39.8, 52.0, 118.5 (q, J=318.0 Hz), 121.3, 128.4, 128.8, 129.8, 144.8,151.0, 167.0. ¹⁹F NMR (CDCl₃, 376 MHz): δ −73.8 (s). FAB-HRMS: Calcd.for C₁₈H₂₂O₅F₃S+[M+H]⁺: 407.1140. found: 407.1133.

4-Cyclooct-2-ynylmethylbenzoic acid methyl ester (8a). To a solution ofvinyl triflate 7a (2.14 g, 5.28 mmol) in THF (30 mL) at 0° C. was addedLDA (3.52 mL, 5.28 mmol, 1.8 M in heptane/THF/ethyl benzene) dropwise bysyringe pump, over 3 h. LDA was added until the starting material wasconsumed. The reaction was quenched with H₂O (10 mL) and the THF wasremoved on a rotary evaporator. The residue was diluted with EtOAc (100mL), washed with H₂O (3×100 mL) and brine (1×50 mL), and dried overMgSO₄. After silica gel chromatography (3:1:0 to 30:10:2hexanes/toluene/EtOAc), a colorless oil was isolated (0.310 g, 23%,R_(f)=0.20 in 30:10:1 hexanes/toluene/EtOAc). IR: 2930, 2857, 1720 cm⁻¹.¹H NMR (CDCl₃, 300 MHz): δ 1.42 (m, 2H), 1.62 (m, 1H), 1.71-1.88 (m,3H), 1.88-1.98 (m, 1H), 2.00-2.10 (m, 1H), 2.11-2.23 (m, 2H), 2.63-2.80(m, 3H), 3.90 (s, 3H), 7.29 (d, 2H, J=8.0 Hz), 7.97 (d, 2H, J=8.4 Hz).¹³C NMR (CDCl₃, 100 MHz): δ 20.8, 28.4, 30.0, 34.8, 36.5, 40.3, 41.7,51.9, 94.9, 96.1, 128.1, 128.9, 129.6, 145.7, 167.1. FAB-HRMS: Calcd.for C₁₇H₂₁O₂ ⁺ [M+H]⁺: 257.1542. found: 257.1536.

4-Cyclooct-2-ynylmethylbenzoic acid (3a). To a solution of cyclooctyne8a (283 mg, 1.11 mmol) in dioxane (12 mL) and H₂O (3 mL) was addedfinely crushed LiOH (530 mg, 22.1 mmol). The suspension was heated to50° C. and stirred for 3 h. The dioxane was removed on a rotaryevaporator. The residue was diluted with EtOAc (50 mL), washed with 1 NHCl (3×50 mL), H₂O (3×50 mL), and brine (2×50 mL), and dried over MgSO₄to yield a white solid (254 mg, 95%, R_(f)=0.25 in 27:3:1hexane/EtOAc/AcOH), mp 112.0-113.9° C. (dec). IR: 3071, 2922, 2846, 1680cm⁻¹. ¹H NMR (CD₃CN, 400 MHz): δ 1.35-1.48 (m, 2H), 1.56-1.66 (m, 1H),1.66-1.95 (m, 4H), 2.01-2.17 (m, 3H), 2.70 (d, 1H, J=5.2 Hz), 2.71 (d,1H, J=1.2 Hz), 2.76 (m, 1H), 7.33 (d, 2H, J=8.4 Hz), 7.91 (d, 2H, J=8.4Hz), 9.40 (br s, 1H). ¹³C NMR (CD₃CN, 100 MHz): δ 21.2, 29.2, 30.8,35.6, 37.3, 40.8, 42.5, 95.7, 97.1, 128.9, 130.1, 130.5, 147.4, 167.8.EI-HRMS: Calcd. for C₁₆H₁₈O₂ M⁺: 242.1307. found: 242.1307. Anal. Calcd.for C₁₆H₁₈O₂: C, 79.31; H, 7.49. Found: C, 79.07; H, 7.26.

4-Cyclooct-2-ynylmethyl)-N-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)propyl]benzamide(12a). TEA (0.184 mL, 0.396 mmol) was added to a solution of cyclooctyne3a (64 mg, 0.26 mmol), amine 13 (106 mg, 0.396 mmol), and HATU (111 mg,0.291 mmol) in CH₂Cl₂ (2 mL) at rt. The reaction was stirred for 15 minat rt, quenched with H₂O (10 mL), and diluted with CH₂Cl₂ (50 mL). Theorganic layer was washed with 1 N HCl (3×50 mL), saturated NaHCO₃ (3×50mL), and brine (2×25 mL), and dried over MgSO₄. Chromatography of thecrude product (2:1 to 1:1 hexanes/EtOAc) yielded the desired product asa colorless oil (69 mg, 69%, R_(f)=0.30 in 1:1 hexanes/EtOAc). IR: 3411,3099, 2925, 2849, 1704, 1639 cm⁻¹. ¹H NMR (CDCl₃, 400 MHz): δ 1.42 (m,2H), 1.61 (m, 1H), 1.70-1.97 (m, 6H), 2.00-2.09 (m, 1H), 2.09-2.22 (m,2H), 2.62-2.78 (m, 3H), 3.38 (q, 2H, J=6.4 Hz), 3.65 (t, 2H, J=6.0 Hz),6.74 (s, 2H), 6.93 (m, 1H), 7.29 (d, 2H, J=8.0 Hz), 7.78 (d, 2H, J=8.0Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 20.8, 28.2, 28.4, 29.9, 34.7, 34.8,36.1, 36.5, 40.0, 41.6, 94.9, 96.2, 126.9, 129.0, 132.2, 134.2, 144.0,167.1, 171.2. FAB-HRMS: Calcd. for C₂₃H₂₇N₂O₃ ⁺ [M+H]⁺: 379.2022. found:379.2014.

Compounds 11b and 11c. Cyclooctyne 3b (0.20 g, 0.77 mmol) and2-azidoethanol (N.J. Agard, unpublished results, 461 μL, 3.84 mmol) weredissolved in CH₃CN (7.2 mL) and Dulbecco's phosphate-buffered saline(1×, pH 7.4, 5.9 mL). The reaction was stirred at rt for 12 h andconcentrated on a rotary evaporator. Chromatography of the crude product(95:4:1 to 90:9:1 CH₂Cl₂/MeOH/AcOH) afforded a 20:1 mixture of 11b:11a(99 mg, 37%, R_(f)=0.30 in 90:9:1 CH₂Cl₂/MeOH/AcOH) and 11c (144 mg,54%, R_(f)=0.15 in 90:9:1 CH₂Cl₂/MeOH/AcOH) as colorless oils.

Compound 11b: mp 175.0-178.0° C. IR: 3442, 2103, 1644 cm⁻¹. ¹H NMR(CD₃OD, 400 MHz): δ 1.39 (m, 3H), 1.55 (m, 1H), 1.68 (m, 1H), 1.79 (m,1H), 2.15 (m, 2H), 2.30 (m, 1H), 2.82 (ddd, 1H, J=14.8, 4.4, 4.4 Hz),3.38 (app t, 1H, J=12.4 Hz), 3.48 (app t, 1H, J=14.4 Hz), 4.02 (m, 1H),4.09 (m, 1H), 4.46 (dt, 1H, J=14.0, 5.2 Hz), 4.62 (ddd, 1H, J=14.0, 8.0,5.6 Hz), 7.05 (d, 2H, J=8.4 Hz), 7.86 (d, 2H, J=8.4 Hz). ¹³C NMR (CD₃OD,100 MHz): δ 22.9, 25.4, 27.4, 28.4, 38.3 (d, J=22.0 Hz), 48.1 (d, J=26.0Hz), 53.9 (d, J=7.0 Hz), 62.1, 97.0 (d, J=174.0 Hz), 130.7, 131.2,132.0, 135.2 (d, J=23.0 Hz), 141.6 (d, J=8.0 Hz), 145.7 (d, J=5.0 Hz),169.8. ¹⁹F NMR (CD₃OD, 376 MHz): δ −145.7 (m). FAB-HRMS: Calcd. forC₁₈H₂₃FN₃O₃ ⁺ [M+H]⁺: 348.1723. found: 348.1722.

Compound 11c: mp 183.0-184.6° C. IR: 3423, 2068, 1643 cm⁻¹. ¹H NMR(CD₃OD, 400 MHz): δ 1.31 (m, 1H), 1.40-1.68 (m, 4H), 1.74 (m, 1H), 1.92(m, 2H), 2.39 (m, 1H), 2.95 (m, 1H), 3.32 (app q, 2H, J=12.8 Hz), 3.88(t, 2H, J=5.6 Hz), 4.34 (m, 2H), 7.05 (d, 2H, J=8.0 Hz), 7.82 (d, 2H,J=8.0 Hz). ¹³C NMR (CD₃OD, 100 MHz): δ 21.7, 23.8, 25.9, 28.1, 38.9,51.1, 51.2, 55.0, 62.2, 130.3, 130.9, 132.0, 135.7, 144.2, 149.9, 170.8.FAB-HRMS: Calcd. for C₁₈H₂₄N₃O₄ ⁺ [M+H]⁺: 346.1767. found: 346.1764.

Kinetic Evaluation of Cyclooctyne Probes

Stock solutions of cyclooctynes 3a and 3b (50 mM), benzyl azide (500 mM)and 2-azidoethanol (500 mM) were made in either CD₃CN or a 55:45 mixtureof CD₃CN and deuterated Dulbecco's phosphate-buffered saline (pH 7.4,made with D₂O). Gentle heating and/or sonication in a H₂O bath wasrequired to fully dissolve 3a and 3b. An NMR tube was charged with 450μL of either 3a or 3b, and 50 μL of either benzyl azide or2-azidoethanol, and the reaction was monitored over time using ¹H NMRspectroscopy.

In the case of the reactions of 3a and 3b with benzyl azide, the kineticdata were derived by monitoring the change in integration of resonancescorresponding to the benzylic protons in benzyl azide (δ ˜4.2 ppm)compared to the corresponding resonances of the triazole products (δ˜5.0 to 5.5 ppm). In the case of the reactions of 3a and 3b with2-azidoethanol, the kinetic data were derived from integration of theresonances corresponding to the more downfield pair of aromatic protons(δ ˜7.9 ppm) in the cyclooctyne starting material compared to thecorresponding resonances in the triazole products (δ ˜7.7 to 7.8 ppm).

Second-order rate constants for the reaction were determined by plotting1/[benzyl azide] versus time in the case of the reactions of 3a and 3bwith benzyl azide and by plotting 1/[3a] versus time or 1/[3b] versustime in the case of the reactions of 3a and 3b with 2-azidoethanol, andsubsequent analysis by linear regression. Second-order rate constantscorrespond to one half of the determined slope (Table 1). Error barsrepresent standard deviations from three replicate experiments.

TABLE 1 Kinetic comparison of azido-ligations^(a). Labeling reagentAzide Solvent k (×10⁻³ M⁻¹s⁻¹) 3b PhCH₂N₃ CD₃CN 4.2 2  PhCH₂N₃ CD₃CN 2.4^(b) 3a PhCH₂N₃ CD₃CN 1.2 1 (R′ = H) PhCH₂N₃ CD₃CN   1.9^(c) 3bHOCH₂CH₂N₃ CD₃CN/PBS (55:45) 4.0 2  HOCH₂CH₂N₃ CD₃CN/PBS (55:45) 2.0^(b) 3a HOCH₂CH₂N₃ CD₃CN/PBS (55:45) 1.1 ^(a)Second-order rateconstants for the [3 + 2] cycloaddition were determined at 22° C. using¹H NMR (see experimental section for a full description of theexperimental setup). ^(b)Agard, N. J., Prescher, J. A., Bertozzi, C. R.J. Am. Chem. Soc. 2004, 126, 15046-15047. ^(c)Lin, F. L., Hoyt, H. M.,van Halbeek, H., Bergman, R. G., Bertozzi, C. R. J. Am. Chem. Soc. 2005,127, 2686-95.Cell Surface Labeling of Jurkat Cells with 12b-FLAG

Jurkat cells (human T-cell lymphoma) were maintained in a 5% CO₂,water-saturated atmosphere and grown in RPMI-1640 media supplementedwith 10% FCS, penicillin (100 units/mL), and streptomycin (0.1 mg/mL).Cell densities were maintained between 1×10⁵ and 1.6×10⁶ cells/mL. Thecells were incubated for 3 d in untreated media or media containing 25μM Ac₄ManNAz. After growth in the presence of Ac₄ManNAz, cells weredistributed into a 96-well V-bottom tissue culture plate. The cells werepelleted (3500 rpm, 3 min) and washed twice with 200 μL of labelingbuffer (PBS, pH 7.4 containing 1% FCS). Cells were then incubated with12b-FLAG or 1-FLAG in labeling buffer for 1 h at rt. After incubation,cells were pelleted, washed twice with labeling buffer, and resuspendedin the same buffer containing FITC-conjugated α-FLAG (1:3000 dilution ofthe Sigma stock). After a 30-min incubation on ice (in the dark), thecells were washed twice with 200 μL of cold labeling buffer and thendiluted to a volume of 400 μL for flow cytometry analysis.

Results

To test the specificity of the cycloaddition reaction in a biologicalcontext, 3a and 3b were further derivatized with FLAG-C (SEQ ID NO:3), ashort peptide epitope for which a fluorescently labeled antibody iscommercially available (Scheme 2). Maleimide derivatives 12a and 12bwere prepared by the HATU-mediated coupling of 3a and 3b withamino-maleimide 13. Cysteine-terminated FLAG peptides were ligated tomaleimides 12a and 12b using standard conditions and the resultingprobes (12a-FLAG and 12b-FLAG) were purified by HPLC for use in celllabeling experiments.

Scheme 2: (a) O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBT), TEA, CH₂Cl₂,69% (12a), 65% (12b); (b) H₂N-DYKDDDDKC-CO₂H (SEQ ID NO:3), DMF/H₂O.

Azides were installed on the surfaces of Jurkat cells by metaboliclabeling of sialic acid residues. Briefly, cells fed peracetylatedN-azidoacetylmannosamine (Ac₄ManNAz), an analog of N-acetylmannosamine(ManNAc), convert the azido sugar to the corresponding azido sialic acid(SiaNAz) within cell surface glycoproteins. Reaction of the cell-surfaceazides with phosphine or cyclooctyne probes containing the FLAG epitopewas monitored by flow cytometry using visualization with aFITC-conjugated α-FLAG antibody (FIG. 4 a).

As shown in FIG. 4 b, the 12b-FLAG labels Jurkat cells in anazide-dependent fashion, similar to a previously studied FLAG-labeledphosphine probe capable of undergoing the Staudinger ligation (1-FLAG).Agard et al. (2004). J. Am. Chem. Soc. 126, 15046-15047. Compound12b-FLAG labels cells in a dose-dependent manner. No toxicity wasobserved in any of these experiments.

FIGS. 4A and 4B. Labeling of cell-surface azides with cyclooctyneprobes. (a) Jurkat cells were incubated with azido sugars for 3 d andthen labeled with various concentrations of 1-FLAG or 12b-FLAG.Detection of the FLAG peptide was achieved using a FITC-conjugatedα-FLAG (anti-FLAG) antibody, followed by analysis by flow cytometry. (b)Dark blue: Cells incubated with 25 μM Ac₄ManNAz for 3 d. Light blue:Cells incubated in the absence of Ac₄ManNAz. Error bars indicate thestandard deviation of three trials. ^(a)Mean fluorescence intensity.

Example 3 Synthesis of an Aryl-Less Cyclooctyne Compound and its Use inLabeling of Living Cells

This example describes synthesis of an “aryl-less” octyne (ALO; shownbelow) and the use of a conjugated ALO to label living cells in vivo.The aryl-less octyne was synthesized; then a maleimide group wasintroduced. The maleimide functionality allows for specific conjugationto the thiol of a cysteine containing peptides. Described below are thesynthesis of the octyne, the octyne-linked maleimides, their conjugationto a FLAG peptide, and the subsequent use of these conjugates on cellsurfaces and in living mice.

Materials and Methods

Synthesis of ALO

Compound 14. AgClO₄ (2.40 g, 11.6 mmol) was added to a solution ofdibromobicycle 1 (1.00 g, 3.72 mmol) and methyl glycolate (6.0 ml, 78.2mmol) dissolved in toluene (4 mL) in a flame-dried,aluminum-foil-wrapped flask. The reaction was stirred for 2 h, dilutedwith pentane (20 mL), and filtered to remove insoluble silver salts. Thesolution was concentrated and purified by silica gel chromatography(5-10% EtOAc: pet ether; R_(f) (10% EtOAc: pet ether)=0.32) to yield 2as a colorless oil (330 mg 1.19 mmol, 22%). ¹H NMR (300 MHz, CD₃Cl) δ6.20 (dd, 1H, J=3.9, 11.7) 4.23 (d, 1H, J=16.5), 4.11 (m, 1H) 3.96 (d,1H, J=16.5), 3.73 (s, 1H), 2.70 (m, 1H), 2.25 (m, 1H), 0.8-2.1 (m, 8H).EI LRMS calculated C₁₁H₁₈O₃Br [M+H]⁺ 278.2 found 278.1.

Compound 15. A suspension of NaOMe (128 mg, 2.38 mmol) in anhydrous DMSO(2 mL) was added to compound 2 (330 mg, 1.19 mmol) dissolved inanhydrous DMSO (3 mL). The reaction was stirred 20 min and additionalNaOMe (250 mg, 4.8 mmol in 1.5 mL of DMSO) was added. The reaction wasstirred until the starting material was completely consumed asdetermined by TLC (20 min). Water (1 ml) was added to the reaction andit was stirred overnight. The reaction was acidified with 1 M HCl (75mL) and extracted twice with EtOAc (50 mL). The combined organicextracts were dried over MgSO₄, filtered, and solvent was removed invacuo. (The reaction can be purified at this point to yield the freeacid, but is most often taken on in crude form to give the activatedpentafluorophenyl ester) The reaction mixture was dissolved in 5 mlCH₂Cl₂, and to this solution was added Et₃N (332 μL, 2.38 mmol) followedby pentafluorophenyltrifluoroacetate (409 μL, 2.38 mmol). The reactionwas stirred 3 h at rt, solvent was removed in vacuo, and the product waspurified by silica gel chromatography (1.5-3% EtOAc: petroleum ethers;R_(f) (3% EtOAc)=0.30) to yield 3 as a clear oil (274 mg, 0.78 mmol,66%). ¹H NMR (400 MHz, CD₃Cl) δ 4.42 (m, 1H), 4.25 (d, 1H, J=15.2), 4.13(d, 1H, J=15.2), 1.5-2.3 (m, 10H) EI LRMS calcd. for C₁₆H₁₄O₃F₅ [M+H]⁺349.3. found 349.0.

Synthesis of Maleimide Octynes

Compounds 16 and 17 were synthesized as follows. TEA (1.5 eq) was addedto a solution of cyclooctyne-PFP (1 eq.), and amine 13 (1.5 eq.), inCH₂Cl₂ at rt. The reaction was stirred for 2 h at rt, quenched with H₂O,and diluted with CH₂Cl₂. The organic layer was washed with 1 N HCl (×3)saturated NaHCO₃ (×3), and brine (×2), and dried over MgSO₄.Chromatography of the crude product (hexanes/EtOAc) yielded the desiredproduct as a colorless oil.

Synthesis of Oct-FLAG Conjugates

Octyne-maleimide (1 eq) was added to a solution of FLAG-C (DYKDDDDKC;SEQ ID NO:3) prepared by standard solid-phase peptide synthesis) (1.2eq) in 1:1 DMF:H₂O and stirred overnight. The crude reaction mixture wasconcentrated and purified via reversed-phase HPLC (Varian Dynamax HPLCsystem with 254-nm detection, on a Microsorb C-18 preparative column ata flow rate of 20 mL/min). All HPLC runs used the following gradient:5-25% acetonitrile in water over 10 min, followed by 35-50% acetonitrilein water over 30 min.

Mice

B6D2F1 mice were obtained from The Jackson Laboratory (Bar Harbor, Me.).All animals were housed and monitored at the Northwest Animal Facility(Berkeley, Calif.), and experiments were performed according toguidelines established by the Animal Care and Use Committee at theUniversity of California, Berkeley (protocol number R234-0504B).

General Protocol for Compound Administration

Mice were administered daily doses of Ac₄ManNAz (0-300 mg/kg in ˜200 μLof 70% DMSO, from a stock solution of 50 mg/mL) intraperitoneally (i.p.)for 7 days. Twenty-four hours after the final Ac₄ManNAz injection, micewere injected i.p. with oct-FLAG (0-40 μmol in ˜200 μL H₂O), Phos-FLAG(20 μmol in ˜200 μL H₂O), or vehicle alone (H₂O). After 3 h, the micewere anesthetized with isoflurane and sacrificed, and their splenocyteswere isolated using a standard protocol.

Labeling of Splenocyte Cell Surface Azides Ex Vivo

Splenocytes from one spleen were suspended in RPMI medium 1640 anddistributed among wells of a 96-well V-bottom tissue culture plate (3wells per treatment, ˜5×10⁵ cells/well). The cells were pelleted (3500rpm, 3 min), rinsed three times with labeling buffer (1% FBS in PBS, pH7.4), and incubated with 0-500 μM oct-FLAG in labeling buffer, Phos-FLAGin labeling buffer, or labeling buffer alone. After incubation at rt for1 h, the cells were rinsed three times with labeling buffer, treatedwith FITC-α-FLAG (1:900 dilution) or FITC-conjugated mouse IgG₁ isotypecontrol (1:200 dilution) in labeling buffer for 30 min on ice, rinsed,and analyzed by flow cytometry.

Results

Splenocyte Labeling of FLAG conjugates: Splenocytes grown in thepresence of Ac₄ManNAz were labeled as described in the procedures. Theresults are shown in FIG. 5. Phosphine-FLAG (“Phos-FLAG”) serves as apositive control of ligation, and the three synthesized octynes all showbioorthogonality as demonstrated by increased labeling in the present ofazide bearing cells. The degree of labeling is consistent with in vitrokinetic data within the octynes. The more hydrophobic octynes(F-oct(MOFO) and Oct (the first reported cyclooctyne) show somebackground.

In vivo labeling of mice: Mice were injected with azide labeled sugarsas described in the procedures. They were subsequently injected withphosphine-FLAG or ALO-FLAG, sacrificed and their cells were analyzed forthe presence of FLAG via flow cytometry. The results are shown in FIG.6. ALO-FLAG, but not F-oct-FLAG or Oct-FLAG, demonstrates the ability toperform this strain-promoted cycloaddition in a living animal.

Example 4 Synthesis and Characterization of Dimethoxy AzacyclooctyneMaterials and Methods

General Synthetic Procedures. All chemical reagents were purchased fromAldrich, Acros, and TCI chemicals and used without purification unlessnoted otherwise. Anhyd DMF and MeOH were purchased from Aldrich or Acrosin sealed bottles; all other solvents were purified via packed columnsas described by Pangborn et al.¹ In all cases, magnesium sulfate wasused as a drying agent and solvent was removed with a Buchi RotovaporR-114 equipped with a Welch self-cleaning dry vacuum. Products werefurther dried on an Edwards RV5 high vacuum. Thin layer chromatographywas performed on Silicycle® 60 Å silica gel plates. Unless otherwisespecified reported R_(f) values are in the solvent system the reactionwas monitored in. Flash chromatography was performed using Merck 60 Å230-400 mesh silica or on a Biotage Flash+® system with Biotage® 10S,10M, 40S or 40M prepacked silica gel columns.

All ¹H and ¹³C NMR spectra are reported in ppm and standardized againstsolvent peaks. All coupling constants (J) are reported in Hz. Spectrawere obtained on Bruker AVB-400®, DRX-500®, or AV-500® instruments. IRspectra were taken on a Varian 3100 FT-IR using thin films on NaClplates. Melting temperatures were obtained on a Barnstead Electrothermal9300 instrument. Optical rotations were measured using a Perkin Elmer241 polarimeter. High resolution fast atom bombardment (FAB) andelectrospray ionization (ESI) mass spectra were obtained from the UCBerkeley Mass Spectrometry Facility. Elemental analysis was performed atthe UC Berkeley Microanalytical Facility.

Methyl 4,6-O-benzylidine-2,3-di-O-methyl-α,D-glucopyranoside (2). Methyl4,6-O-benzylidene-α,D-glucopyranoside (1.416 g, 5.176 mmol, Acros) wasdissolved in toluene (55 mL, anhyd). To this solution, KOH was added(1.73 g, 30.8 mmol, 6 equiv) followed by CH₃I (2.20 mL, 35.3 mmol, 7equiv). The mixture was heated to reflux while stirring under N₂ andmonitored by TLC (1:1 hexanes/EtOAc) for the disappearance of 1(R_(f)=0.2). Upon reaction completion (approx 4 h), the mixture wascooled to rt and toluene (50 mL) was added and washed with H₂O (3×30mL). The toluene was dried, decanted, evaporated to dryness and twiceazeotroped with toluene to result in 2 as a white powder (1.516 g, 4.885mmol, 94%, R_(f)=0.7). Mp 123.2-124.0° C. (lit.² 121-123° C.). ¹H NMR(400 MHz, CDCl₃): δ 7.48-7.46 (m, 2H), 7.34-7.28 (m, 3H), 5.51 (s, 1H),4.82 (d, J=3.4 Hz, 1H), 4.25 (dd, J=9.9, 4.5 Hz, 1H), 3.79 (td, 5.1, 4.4Hz, 1H), 3.70 (t, J=10.1 Hz, 1H), 3.66 (t, J=9.2 Hz, 1H), 3.60 (s, 3H),3.52 (s, 3H), 3.50 (t, J=9.3 Hz, 1H), 3.41 (s, 3H), 3.26 (dd, J=9.2, 3.7Hz, 1H). ¹³C NMR (100 MHz, CDCl₃): δ 137.4, 129.0, 128.3, 126.1, 101.4,98.4, 82.2, 81.5, 79.9, 69.1, 62.3, 61.1, 59.4, 55.3. HRMS (FAB) calcdfor C₁₆H₂₃O₆ [M+H]⁺: 311.149464. found: 311.14930.

Methyl 6-bromo-6-deoxy-2,3-di-O-methyl-α,D-glucopyranoside (3). Methyl4,6-O-benzylidene-α,D-glucopyranoside (37.97 g, 122.4 mmol) wasdissolved in CCl₄ (1.5 L, anhyd) and CaCO₃ (13.54 g, 135.3 mmol, 1.11equiv) was added. This mixture was heated to reflux under N₂. Oncereaching reflux, N-bromosuccinimide (24.228 g, 136.13 mmol, 1.11 equiv,recrystallized) was added and the reaction was monitored by TLC (1:1hexanes/EtOAc) for the dissapearance of 2 (R_(f)=0.7). Upon reactioncompletion (approx 1 h), it was cooled to rt and evaporated to dryness.The residue was dissolved in CH₂Cl₂ (1 L) and washed with 10% Na₂SO₃(1×1 L), sat. NaHCO₃ (1×1 L). Each aqueous wash was extracted withCH₂Cl₂ (2×500 mL). All organic layers were combined, dried, decanted,and evaporated to dryness. The residue was dissolved in a solution of 1%NaOH in MeOH (1.5 L). After 1 h, the solution was neutralized with 3MHCl and evaporated to dryness. The residue was dissolved in H₂O (1.5 L)and extracted with CH₂Cl₂ (8×500 mL). The organic layers were combined,dried, decanted, and evaporated to dryness. The crude product waspurified on 5 Biotage 40M columns with a gradient solvent system of 4:1hexanes/EtOAc to 1:1 hexanes/EtOAc to result in pure 3 as a clear oil(30.10 g, 105.6 mmol, 86%, R_(f)=0.3). ¹H NMR (400 MHz, CDCl₃): δ 4.73(d, J=3.5 Hz, 1H), 3.60-3.53 (m, 3H), 3.49 (s, 3H), 3.49-3.43 (m, 1H),3.35-3.24 (m, 8H), 3.11 (dd, J=9.1, 3.5 Hz, 1H). ¹³C NMR (100 MHz,CDCl₃): δ 97.3, 82.7, 81.7, 71.6, 69.9, 61.2, 58.5, 55.3, 33.5. HRMS(FAB) calcd for C₉H₁₇BrO₅Li [M+Li]⁺: 291.041939. found: 291.041570.

(2S,3S,4R)N-allyl, N-(methyl succinyl)-4-hydroxy-2,3-dimethoxyhex-5-eneamine (4). Pyranoside 3 (18.2 g, 63.8 mmol) was dissolved in 19:11-propanol/H₂O (1.5 L) in an Erlenmeyer flask equipped with an overheadstirring unit. To this solution, allylamine (150 mL, 2.0 mol, 31 equiv),zinc (223.9 g, 3.423 mol, 54 equiv, acid treated), and NaBH₃CN (18.97 g,301.9 mmol, 5 equiv) were added. The reaction was heated to 90° C. andmonitored by TLC (EtOAc) for the disappearance of 3 (R_(f)=0.7). Uponreaction completion (approx 1 h), the mixture was cooled to rt andfiltered through Celite. The filtrate was evaporated to dryness anddissolved in 6:4:1 MeOH/CH₂Cl₂/1.5M HCl (1.32 L) and stirred for 1 h(adding 3M HCl as necessary to keep the solution acidic throughout theh) at which point, H₂O (300 mL) was added and the mixture was extractedwith CH₂Cl₂ (3×600 mL). The organics were dried, decanted, andevaporated to a residue. The resulting crude amine was dissolved in MeOH(600 mL, anhyd). To this solution, N,N-diisopropylethylamine (12.2 mL,70.0 mmol, 1.1 equiv) followed by methyl succinyl chloride (8.6 mL, 70mmol, 1.1 equiv) were added and the mixture was stirred at rt under N₂for 1 h, at which point the reaction was quenched with H₂O (100 mL) andthe MeOH was removed via rotary evaporation. To the resulting aqueoussolution, H₂O (500 mL) was added and extracted with CH₂Cl₂ (3×650 mL).The organics were combined, dried, decanted, and evaporated to dryness.The crude product was purified on 3 Biotage 40M columns with a gradientsolvent system starting with 25:1 toluene/acetone and ending with 3:1toluene/acetone (product begins to elute at 10:1 toluene/acetone) toresult in pure 4 as a colorless oil (14.776 g, 44.858 mmol, 70%).R_(f)=0.6 in 1:1 toluene/acetone. [α]_(D) ²⁸ −38.8° (c 0.943, CH₂Cl₂).1:0.5 mixture of rotamers (designated rot) ¹H NMR (500 MHz, CDCl₃): δ5.98-5.88 (m, 1H, 1rotH), 5.79-5.71 (m, 1H, 1rotH), 5.36 (d, J=17.2 Hz,1rotH), 5.35 (d, J=17.2 Hz, 1H), 5.24-5.11 (m, 3H, 3rotH), 4.32-4.27 (m,1H, 1rotH), 4.16-4.13 (m, 1H, 1rotH), 4.07-4.03 (m, 1H), 3.98-3.94 (m,1rotH), 3.78 (dd, J=13.9, 3.6 Hz, 1H), 3.72-3.67 (m, 4H, 3rotH),3.63-3.59 (m, 2rotH), 3.52 (s, 3H, 3 rotH), 3.45-3.38 (m, 3H, 4rotH),3.25-3.21 (m, 1H, 1rotH), 3.15 (t, J=4.2 Hz, 1H), 2.85-2.80 (m, 1rotH),2.73-2.60 (m, 5H, 3rotH), 2.33 (d, J=6.5 Hz, 1rotH). ¹³C NMR (125 MHz,CDCl₃): δ 173.9, 173.7, 172.2, 172.1, 138.4, 133.6, 132.8, 117.2, 116.7,116.31, 116.25, 83.4, 82.7, 80.5, 80.4, 72.4, 71.5, 60.7, 60.6, 59.8,59.7, 52.02, 51.95, 51.9, 48.9, 48.3, 48.2, 29.5, 29.2, 28.2, 28.1. IR:3441 (b), 3082, 2981, 2933, 2832, 1737, 1641 cm⁻¹. HRMS (FAB) calcd forC₁₆H₂₈NO₆[M+H]⁺: 330.191663. found: 330.192190. Anal. calcd forC₁₆H₂₇NO₆: C, 58.34; H, 8.26; N, 4.25. found: C, 58.41; H, 8.22; N,4.38.

(5R,6S,7S,Z)N-(methyl succinyl)-5-hydroxy-6,7-dimethoxyazacyclooct-3-ene(5). Compound 4 (790 mg, 2.40 mmol) was dissolved in CH₂Cl₂ (200 mL,anhyd) and heated to reflux while stirring under N₂. Once at reflux,Grubbs second generation catalyst (163.3 mg, 0.1927 mmol, 0.08 equiv)was added and the reaction was carefully monitored by TLC in 1:1toluene/acetone for the disappearance of # (R_(f)=0.6) adding morecatalyst if necessary (at 2.25 h 61 mg catalyst added). Upon completion(approx 5 h), the mixture was cooled to rt, evaporated to dryness, andimmediately purified on a Biotage 40M column with a gradient of 8:1toluene/acetone, 6:1 toluene/acetone, 4:1 toluene/acetone. Thisprocedure resulted in pure 5 as a brown oil (500.1 mg, 1.661 mmol, 69%,R_(f)=0.4). [α]_(D) ²⁸ −82.6° (c 1.12, CH₂Cl₂). 1:0.18 mixture ofrotamers. ¹H NMR (500 MHz, CDCl₃): δ 5.63 (ddd, J=11.9, 6.4, 2.0 Hz,1H), 5.56-5.54 (m, 2rotH), 5.48-5.45 (m, 1H), 4.41-4.30 (m, 2rotH), 4.35(t, J=8.0 Hz, 1H), 4.30 (apparent d, J=17.5 Hz, 1H), 4.07 (dd, J=13.8,3.1 Hz, 1H), 3.73 (dd, J=17.2, 5.0 Hz, 1H, 2rotH), 3.66 (s, 3H, 3rotH),3.62-3.56 (m, 4H, 3rotH), 3.50 (s, 3H), 3.45 (s, 3rotH), 3.42-3.39 (m,2rotH), 3.27 (s, 1H), 3.08 (apparent dd, J=9.0 Hz, 5.6 Hz, 1rotH), 2.93(dd, J=9.4, 7.7 Hz, 1H, 1rotH), 2.81 (dd, J=13.8, 9.6 Hz, 1H), 2.68-2.52(m, 4H, 4rotH). ¹³C NMR (100 MHz, CDCl₃): δ 173.3, 173.1, 171.5, 134.5,132.4, 125.3, 123.7, 85.5, 85.2, 81.4, 80.5, 68.1, 66.8, 60.8, 60.1,58.0, 57.8, 51.6, 47.8, 46.0, 45.3, 45.2, 29.0, 28.7, 28.3. IR: 3472(b), 2933, 2828, 1735, 1636 cm¹. HRMS (FAB) calcd for C₁₄H₂₄NO₆ [M+H]⁺:302.160363. found: 302.159780.

(6R,7S,Z)N-(methyl succinyl)-6,7-dimethoxy-5-oxoazacylclooct-3-ene. To asolution of 5 (435.6 mg, 1.505 mmol) in CH₂Cl₂ (100 mL, anhyd),pyridinium chlorochromate (494.4 mg, 2.294 mmol, 1.5 equiv) was added.The mixture was heated to 40° C. and stirred under N₂ overnight. Thefollowing day, the reaction was cooled to rt, H₂O (75 mL) was added, andextracted with CH₂Cl₂ (3×100 mL). The organics were combined, dried,decanted, and evaporated to dryness to result in crude product which waspurified on a Biotage 40S column using a solvent system of 8:1toluene/acetone, 6:1 toluene/acetone, 4.5:1 toluene/acetone to result inpure (6R,7S,Z)N-(methyl succinyl)-6,7-dimethoxy-5-oxoazacylclooct-3-eneas a clear oil (360 mg, 1.21 mmol, 80%). R_(f)=0.6 in 1:1toluene/acetone. [α]_(D) ²⁸ +35.5° (c 4.46, CH₂Cl₂). 1:1 mixture ofrotamers. ¹H NMR (500 MHz, CDCl₃): δ 6.17 (apparent d, J=11.8 Hz, 1H),5.95-5.88 (m, 2H), 5.69 (d, J=11.9 Hz, 1H), 4.26 (dd, J=17.4, 4.3 Hz,1H), 4.11 (d, J=19.7 Hz, 1H), 3.96 (d, J=19.7 Hz, 1H), 3.81-3.71 (m,6H), 3.61-3.59 (m, 8H), 3.48-3.30 (m, 13H), 2.62-2.40 (m, 7H), 2.36-2.31(m, 1H). ¹³C NMR (125 MHz, CDCl₃): δ 203.2, 202.2, 173.8, 173.7, 172.5,171.9, 137.5, 132.5, 129.1, 126.3, 89.1, 87.3, 81.1, 79.3, 59.6, 59.09,59.07, 58.3, 51.9, 51.8, 50.4, 49.7, 48.5, 46.8, 29.1, 29.0, 28.02,27.95. IR: 3590, 3516, 2944, 2830, 1743, 1691, 1655 cm⁻¹. HRMS (FAB)calcd for C₁₄H₂₂NO₆ [M+H]⁺: 300.144713. found: 300.144130. Anal. calcdfor C₁₄H₂₁NO₆: C, 56.18; H, 7.07; N, 4.68. found: C, 56.17; H, 7.09; N,4.57.

(3S,4R)N-(methyl succinyl)-3,4-dimethoxy-5-oxoazacyclooctane (6).(6R,7S,Z)N-(methyl succinyl)-6,7-dimethoxy-5-oxoazacylclooct-3-ene(330.9 mg, 1.109 mmol) was dissolved in EtOH (60 mL) and 10% Pd/C (27.8mg) was added. The mixture was stirred overnight at rt under H₂. Thefollowing day, the mixture was filtered through Celite and the filtratewas evaporated to dryness to yield 6 (295 mg, 0.980 mmol, 89%).R_(f)=0.5 in 1:1 toluene/acetone. [α]_(D) ²⁸ +29.7° (c 6.24, CH₂Cl₂).1:0.66 mixture of rotamers. ¹H NMR (500 MHz, CDCl₃): δ 4.00 (dd, J=14.0,4.5 Hz, 1H), 3.87 (d, J=8.0 Hz, 1H), 3.75-3.69 (m, 3rotH), 3.65-3.39 (m,8H, 6rotH), 3.34 (s, 3rotH), 3.29-3.20 (m, 3H, 2rotH), 3.08 (dt, J=14.5,5.0 Hz, 1H), 2.69-2.25 (m, 6H, 8rotH), 2.12-2.07 (m, 3H), 1.96 (bs,1rotH). ¹³C NMR (125 MHz, CDCl₃): δ 209.7, 209.0, 173.8, 173.5, 172.8,172.4, 88.4, 86.1, 82.3, 80.4, 59.8, 59.1, 58.8, 57.9, 51.9, 51.8, 49.4,48.3, 47.8, 47.5, 41.3, 37.7, 29.2, 28.9, 28.5, 28.3, 26.0, 25.3. IR:3587, 3518, 2940, 2831, 1743, 1711, 1655 cm¹. HRMS (ESI) calcd forC₁₄H₂₃NO₆Na [M+Na]⁺: 324.1418. found: 324.1420. Anal. calcd forC₁₄H₂₃NO₆: C, 55.80; H, 7.69; N, 4.65. found: C, 55.91; H, 7.77; N,4.63.

Compound 7. Ketone 6 (120.2 mg, 0.3993 mmol) was dissolved in 1:1H₂O/EtOH containing 100 mM aniline (4 mL). To this solution,semicarbazide hydrochloride (463.3 mg, 4.136 mmol, 10 equiv) and AcOH(55 drops) was added. The reaction was stirred at rt and monitored byTLC (1:1 toluene/acetone) for the disappearance of 6 (R_(f)=0.5). Afterthe reaction was complete (approx 8 h), the reaction was evaporated todryness. The crude white solid was sonicated with EtOAc (4×5 mL). TheEtOAc was combined, dried, decanted, and evaporated to dryness to resultin crude semicarbazone that was directly converted to selenadiazole 7.The crude semicarbazone was dissolved in dioxane (0.8 mL). A solution ofSeO₂ (204.1 mg, 1.839 mmol, 5 equiv) in 1:1 dioxane/H₂O (0.6 mL) wasadded to the semicarbazone solution. The mixture was stirred overnightat rt and analyzed the following day by LCMS for the presence of 7([M+H]⁺=392) and absence of semicarbazone ([M+H]⁺=359, [M+Na]⁺=381).Additional SeO₂ was added if necessary to force the reaction tocompletion (128.3 mg added). Upon reaction completion, the dioxane wasevaporated off and more H₂O (10 mL) was added. The aqueous solution wasextracted with EtOAc (3×25 mL) and the organics were combined, dried,decanted, and evaporated to dryness. The crude product waschromatographed on a Biotage 40S column with CH₂Cl₂, 80:1 CH₂Cl₂/MeOH,70:1 CH₂Cl₂/MeOH to result in pure 7 as a yellow oil (68 mg, 0.17 mmol,43%). R_(f)=0.3 in 60:1 CH₂Cl₂/MeOH. [α]_(D) ²⁸ +17.6° (c 1.27, CH₂Cl₂).1:0.8 mixture of rotamers. ¹H NMR (500 MHz, CDCl₃): δ 5.39 (s, 1rotH),5.37 (d, J=5.4 Hz, 1H), 4.36 (m, 1rotH), 4.07-4.02 (m, 2rotH), 3.91 (q,J=5.3 Hz, 1H), 3.73 (bs, 2H), 3.67 (s, 3rotH), 3.65 (s, 3H), 3.59-3.53(m, 4H, 3rotH), 3.45-3.39 (m, 2H, 2rotH), 3.33-3.19 (m, 3H, 5rotH),2.91-2.83 (m, 1rotH), 2.74-2.53 (m, 5H, 1rotH), 2.48-2.36 (m, 2rotH).¹³C NMR (125 MHz, CDCl₃): δ 173.9, 173.7, 173.4, 171.7, 161.2, 159.3,157.0, 156.3, 82.6, 80.1, 78.8, 77.9, 58.7, 58.5, 57.94, 57.88, 52.0,51.9, 51.3, 50.5, 50.1, 49.8, 29.6, 29.1, 28.4, 28.3, 26.3, 25.4. IR:3580, 3057, 2983, 2931, 2828, 1741, 1649 cm⁻¹. HRMS (FAB) calcd forC₁₄H₂₂N₃O₅Se [M+H]⁺: 392.072467. found: 392.071460.

(6S,7S)N-(methyl succinyl)-6,7-dimethoxyazacyclooct-4-yne. Selenadiazole7 (83.5 mg, 0.214 mmol) was dissolved in m-xylene (50 mL) and heated to115° C. The reaction was monitored by TLC (1:1 toluene/acetone) for thedisappearance of 7 (R_(f)=0.60, UV active, red spot with vanillin) andappearance of azacyclooctyne methyl ester (R_(f)=0.65, green spot withvanillin). Upon reaction completions (approx 30 h), it was cooled to rt,filtered, and the filtrate was evaporated to dryness. The crude productwas purified on silica gel (9.5 in³) using a toluene/acetone solventsystem starting at 20:1 and ending with 8:1. This procedure resulted inpure (6S,7S)N-(methyl succinyl)-6,7-dimethoxyazacyclooct-4-yne as aslightly yellow oil (27.3 mg, 0.0964 mmol, 45%, R_(f)=0.6). [α]_(D) ²⁸+7.5° (c 0.64, CH₂Cl₂). 1:0.15 mixture of rotamers. ¹H NMR (400 MHz,D₂O): δ 4.38 (apparent d, J=7.9 Hz, 1rotH), 4.24 (dt, J=8.6, 2.6 Hz, 1H,1rotH), 4.17 (dd, 14.9, 5.4 Hz, 1H), 4.06 (d, J=14.3 Hz, 1H), 4.00 (s,1rotH), 3.88 (t, J=9.2 Hz, 1rotH), 3.72-3.68 (m, 4H, 3rotH), 3.55-3.44(m, 3H, 4rotH), 3.38-3.29 (m, 4H, 3rotH), 3.05 (dd, J=14.3, 9.1 Hz, 1H,1rotH), 2.90-2.64 (m, 5H, 5rotH), 2.33 (dt, J=16.9, 3.2 Hz, 1H), 2.25(apparent d, J=16.8 Hz, 1rotH). ¹³C NMR (100 MHz, CDCl₃): δ 173.6(broad), 172.5, 171.6, 99.0, 96.0, 93.0, 91.0, 87.0, 85.2, 77.8, 77.4,59.8, 59.3, 57.5, 57.4, 56.4, 55.7, 53.2, 52.3, 52.02, 51.98, 29.5,29.3, 28.7, 28.3, 22.1, 21.1. IR: 3489, 2931, 2827, 2203, 1736, 1648cm⁻¹. HRMS (FAB) calcd for C₁₄H₂₂NO₅ [M+H]⁺: 284.149798. found:284.150650.

(6S,7S)N-(succinic acid)-6,7-dimethoxyazacyclooct-4-yne (8). Cyclooctynemethyl ester (27.3 mg, 0.0964 mmol) was dissolved in 2:1 H₂O/dioxane(1.5 mL) and LiOH (45.7 mg, 1.91 mmol, 20 equiv, crushed) was added. Thereaction was stirred overnight at rt. The following day the mixture wasneutralized with 3M HCl and the dioxane was evaporated off. AdditionalH₂O (3 mL) was added to the resulting aqueous solution, this solutionwas acidified with 3M HCl and extracted with EtOAc (5×10 mL). The EtOAcwas combined, dried, decanted, and evaporated to dryness. The crudeproduct was purified on silica gel using a gradient solvent systemstarting with 8:1 toluene/acetone and ending with 1:1 toluene/acetone.This procedure resulted in pure 8 as an off white solid (17.1 mg, 0.0636mmol, 66%). R_(f)=0.3−0.4 in 1:1 toluene/acetone. [α]_(D) ²⁸ −14.6° (c0.357, H₂O). 1:0.1 mixture of rotamers. ¹H NMR (400 MHz, D₂O): δ 4.37(dt, J=7.8, 2.3 Hz, 1rotH), 4.13 (dt, J=8.7, 2.8 Hz, 1H, 1rotH), 4.18(dd, J=14.9, 5.4 Hz, 1H), 4.06 (d, J=14.3 Hz, 1H), 3.91 (s, 1rotH), 3.89(t, J=8.4 Hz, 1rotH), 3.71 (t, J=8.5 Hz, 1H), 3.56 (s, 3H, 3rotH),3.56-3.46 (m, 2rotH), 3.37-3.28 (m, 4H, 2rotH), 3.04 (dd, J=14.3, 9.0Hz, 1H, 1rotH), 2.88-2.64 (m, 5H, 5rotH), 2.32 (dt, J=16.6, 3.4 Hz, 1H),2.24 (apparent d, 16.8 Hz, 1rotH). ¹³C NMR (125 MHz, D₂O, no rotamerpeaks tabulated): δ 177.0, 174.9, 99.0, 89.7, 84.1, 76.1, 58.0, 56.4,54.1, 51.9, 29.0, 27.7, 20.6. IR: 3434, 2935, 2830, 2358, 2207, 1729,1642 cm¹. HRMS (ESI) calcd for C₁₃H₁₉NO₅Na [M+Na]⁺: 292.1155. found:292.1157.

Azacyclooctyne biotin conjugate (9). Cyclooctyne free acid 8 (4.8 mg,0.018 mmol) was dissolved in CH₃CN (1 mL, anhyd) and cooled to 0° C.N,N-Diisopropylethylamine (10 μL, 0.06, 3 equiv) was added and thissolution was stirred under N₂ for 10 min at which point,pentafluorophenyl trifluoroacetate (10 μL, 0.058 mmol, 3 equiv) wasadded dropwise and the reaction was allowed to warm to rt. The r×n wasmonitored by TLC (1:1 toluene/acetone) for the disappearance of 8(R_(f)=0.3-0.4). Upon reaction completion (approx 1 h), the mixture wasfiltered, and the filtrate was evaporated to dryness. Thepentafluorophenyl activated cyclooctyne was purified on silica gel usinganhyd toluene and anhyd ether in a gradient solvent system of 10:1toluene/ether to 4:1 toluene/ether. This product was dried andimmediately used for the coupling to biotin.N-(13-amino-4,7,10-trioxamidecanyl)biotinamide³ (7.8 mg, 0.018 mmol, 1equiv) was dissolved in DMF (0.5 mL, anhyd) and cooled to 0° C.N,N-Diisopropylethylamine (2 drops) was added. The pentafluorophenylactivated cyclooctyne was dissolved in DMF (0.5 mL, anhyd) and thissolution was added dropwise to the biotin solution at 0° C. Uponaddition of all activated cyclooctyne, the reaction was warmed to rt andmonitored by ESI-LCMS for the formation of 9 ([M+H]⁺=698, [M+Na]⁺=720).Upon reaction completion (approx 6 h), the mixture was evaporated todryness and purified by flash chromatography on silica gel. A gradientsolvent system was used beginning at 50:3:1 EtOAc/MeOH/H₂O and endingwith 8:3:1 EtOAc/MeOH/H₂O. This procedure resulted in pure 9 (5.0 mg,0.0072 mmol, 40%). R_(f)=0.4 in 5:3:1 EtOAc/MeOH/H₂O. ¹H NMR (500 MHz,D₂O): δ 4.58 (dd, J=7.9, 4.9 Hz, 1H), 4.40 (dd, J=7.9, 4.5 Hz, 1H), 4.36(dt, J=7.8, 2.0 Hz, 0.1H), 4.22 (dt, J=8.6, 2.5 Hz, 1H), 4.12 (dd, 14.9,5.4 Hz, 0.9H), 4.05 (d, J=14.2 Hz, 0.9H), 4.00 (d, J=16.2 Hz, 0.1H),3.82 (t, J=8.4 Hz, 0.1H), 3.71-3.65 (m, 8.9H), 3.57-3.45 (m, 7.1H),3.37-3.19 (m, 8.9H), 3.03 (dd, J=14.3, 9.0 Hz, 1H), 2.97 (dd, J=13.1,5.0 Hz, 1H), 2.92-2.88 (m, 0.1H), 2.79-2.75 (m, 2.9H), 2.65-2.62 (m,1H), 2.58-2.49 (m, 2H), 2.32 (dt, J=16.8, 3.0 Hz, 0.9H), 2.24 (t, J=7.2Hz, 2.1H), 1.79-1.53 (m, 8H), 1.44-1.33 (m, 2H). ¹³C NMR (125 MHz, D₂O):δ 176.7, 174.7, 174.5, 165.2, 98.9, 89.9, 84.3, 76.1, 69.5, 69.3, 68.4,68.3, 62.0, 60.2, 58.0, 56.5, 55.3, 54.2, 52.0, 39.6, 36.3, 36.2, 35.4,30.7, 28.24, 28.18, 27.8, 27.6, 25.1, 20.8. HRMS (ESI) calcd forC₃₃H₅₅N₅O₉SNa [M+Na]⁺: 720.3618. found: 720.3593.

Scheme 3, below, depicts the synthesis of azacyclooctyne 8 and biotinconjugate 9.

Determination of Rate Constant for the Reaction of Azacyclooctyne (8)and Benzyl Azide.

The reaction in Scheme 4 was monitored by ¹H NMR for 4 h. Azacyclooctyne8 and benzyl azide were separately dissolved in CD₃CN and mixed togetherat a 1:1 concentration of 26 mM. Tert-butyl acetate was used as aninternal standard. The percent conversion was calculated by thedisappearance of azacyclooctyne relative to the tert-butyl acetate, asdetermined by integration. No products other than 10 and 11 wereapparent by ¹H NMR. The second order rate constant was determined byplotting 1/[8] versus time. The plot was fit to a linear regression andthe slope corresponds to the second order rate constant.

Cell Culture Procedures

Jurkat cells (human T-cell lymphoma) were grown in RPMI-1640 media(Invitrogen Life Technologies) that was supplemented with 10% fetal calfserum (FCS, Hyclone), penicillin (100 units/mL), and streptomycin (0.1mg/mL). The cells were maintained in a 5% CO₂ water-saturated atmosphereand their media was changed every 3 d keeping the cells at densitiesbetween 1×10⁵ and 1.6×10⁶ cells/mL (as determined using a Coulter Z2cell counter).

Western Blot Analysis of Azide-Labeled Cell Lysates

Jurkat cells were grown with or without 25 μM Ac₄ManNAz (+ or −respectively) for 3 d as described above. The cells were pelleted (3500rpm, 4 min) and washed with chilled PBS (3×10 mL). The pellet wassuspended in lysis buffer (150 mM NaCl, 20 mM Tris, 1% NP40, pH 7.4containing mini-protease inhibitors; 2 mL lysis buffer/1 L jurkatlysate) and sonicated (3×30 sec). Following sonication, the lysed cellswere pelleted (3700 g for 30 min) and the supernatant was kept. ABio-RAD® D_(c) protein assay was performed to determine the proteinconcentration of each lysate.

150 μG of protein from each lysate was treated with 250 mM 9 or noreagent overnight. 4×SDS-PAGE loading buffer was added to each sampleand the samples were separated via electrophoresis and thenelectroblotted to a nitrocellulose membrane. The membrane was blockedusing 5-10% BSA in PBST (PBS pH 7.4 containing 0.1% Tween 20) for 2 h atrt. The blot was incubated with a horse radish peroxidase-conjugatedanti-biotin antibody (HRP-α-biotin) (1:100,000) in PBST for 1 h. Themembrane was washed with PBST (3×15 min). Detection was performed bychemiluminescence using Pierce SuperSignal® West Pico ChemiluminescentSubstrate.

The data are shown in FIG. 7. FIG. 7 presents a Western blot of Jurkatcells lysate treated with (⁺Az) or without (⁻Az) Ac₄ManNAz and labeledwith 0 or with 250 μM azacycloocytne biotin conjugate 9. Jurkat cellswere treated with 0 (−Az) or 25 μM (+Az) Ac₄ManNAz for 3 days and lysed.Lysates were reacted with an azacyclooctyne-biotin conjugate (250 μM) orno reagent, and analyzed by SDS-PAGE. A Western blot was performed usinganti-biotin antibody. Numbers indicate apparent molecular weight, andequal protein loading was confirmed using Ponceau S stain. The datapresented in FIG. 7 demonstrate that azacyclooctyne-biotin canselectively label azide-containing glycoproteins in a cell lysate.

Cell Surface Azide Labeling And Detection.

Jurkat cells were incubated in untreated media or media containing 25 μMAc₄ManNAz. After 3 d, the cells were pelleted are resuspended in FACSbuffer (PBS containing 1% FCS, 2×10 mL) and approximately 500,000 cellswere placed in each well of a 96 well V-bottom plate. The cells werepelleted (3500 rpm, 3 min) and washed with FACS buffer (1×200 μL). Cellswere then incubated for 1 h at rt with 9 or 12 at 250 μM in FACS bufferwith 3% DMF (100 μL) or FACS buffer with 3% DMF and no reagent (100 μL).Upon completion of the incubation, cells were pelleted and washed withcold FACS buffer (3×200 μL). Cells were resuspended in FACS buffer (100μL) containing FITC-avidin (1:200 dilution of Sigma stock) and incubatedin the dark at 0° C. for 15 min. Following the incubation, cells werepelleted, washed with cold FACs buffer (1×200 μL) and anotherFITC-avidin incubation was preformed. After the final, FITC-avidinlabeling, the cells were washed with cold FACS buffer (3×200 μL) andthen diluted in FACS buffer (400 μL) for flow cytometry analysis. Flowcytometry was performed on a BD Biosciences FACSCalibur flow cytometerequipped with a 488-nm argon laser. All flow cytometry experiments wereperformed in triplicate, and 20,000 cells were collected per a sample.

The data are shown in FIG. 8. FIG. 8 presents data showing cell surfacelabeling of Jurkat cells grown in the presence (Az) or absence (no Az)of Ac₄ManNAz for 3 days. Cells were labeled for 1 hour with no reagent,with azacyclooocytne biotin conjugate 9 (250 μM), or with a cycloocytynebiotin conjugate 12 (250 μM).

The cells were then stained with fluorescein isothiocyanate(FITC)-labeled avidin and analyzed by flow cytometry. Shown is the meanfluorescence intensity (MFI, in arbitrary units). Error bars representtriplicate samples from the same experiment. Cyclooctyne biotin 12 is abiotin conjugate of the aryl-less octyne (“ALO”)

The data presented in FIG. 8 demonstrate that azacyclooctyne-biotin canlabel azide-containing membrane-associated glycans in live cells.

Example 5 Synthesis and Characterization of Difluorinated CycloocytnesGeneral Materials and Methods

All chemical reagents were of analytical grade, obtained from commercialsuppliers, and used without further purification unless otherwise noted.With the exception of reactions performed in aqueous media, all reactionvessels were flame-dried prior to use. Reactions were performed in a N₂atmosphere, except in the case of reactions performed in aqueous media,and liquid reagents were added with a syringe unless otherwise noted.Tetrahydrofuran (THF) was distilled under N₂ from Na/benzophenoneimmediately prior to use, and CH₂Cl₂ was distilled from CaH₂ immediatelyprior to use. Flash chromatography was carried out with Merck 60 230-400mesh silica gel according to the procedure described by Still (1).Reactions and chromatography fractions were analyzed with Analtech 250micron silica gel G plates and visualized by staining with cericammonium molybdate, anisaldehyde, or by absorbance of UV light at 245nm. Organic extracts were dried over MgSO₄, and solvents were removedwith a rotary evaporator at reduced pressure (20 torr), unless otherwisenoted. Unless otherwise noted, ¹H, ¹³C, and ¹⁹F NMR spectra wereobtained with 300 MHz or 400 MHz Bruker spectrometers. Chemical shiftsare reported in δ ppm referenced to the solvent peak for ¹H and ¹³C andrelative to CFCl₃ for ¹⁹F. Coupling constants (J) are reported in Hz.Low- and high-resolution fast atom bombardment (FAB) and electron impact(EI) mass spectra were obtained at the UC Berkeley Mass SpectrometryFacility, and FT-ICR mass spectra were obtained at the Howard HughesMedical Institute Mass Spectrometry Facility at UC Berkeley.Reversed-phase HPLC was performed using a Rainin Dynamax SD-200 HPLCsystem with 210 nm detection on a Microsorb C18 analytical orpreparative column.

Dulbecco's phosphate-buffered saline (PBS), fluorescein isothiocyanate(FITC)-a-FLAG, and bovine serum albumin (BSA) were purchased from Sigma.RPMI-1640 media was obtained from Invitrogen Life Technologies, Inc.,and fetal bovine serum (FBS) was purchased from HyClone Laboratory.FITC-conjugated mouse IgG₁ isotype control was obtained from BDPharmingen. Flow cytometry analysis was performed on a BD FACSCaliburflow cytometer using a 488 nm argon laser. At least 10⁴ cells wereanalyzed for each sample. Cell viability was ascertained by gating thesamples on the basis of forward scatter (to sort by size) and sidescatter (to sort by granularity). The average fluorescence intensity wascalculated from each of three replicate experiments to obtain arepresentative value in arbitrary units. For all flow cytometryexperiments, data points were collected in triplicate and arerepresentative of at least three separate experiments.

Synthetic Procedures

The synthesis of DIFO (1) was carried out as outlined in Schemes 5 and6. Various derivatives of DIFO containing fluorophores (DIFO-488,DIFO-568, and DIFO-647), biotin (DIFO-biotin), and the FLAG peptide(DIFO-FLAG) were synthesized as outlined in Scheme 7.

Monoallylated cyclooctanediol (7). To a solution ofcis-1,5-cyclooctanediol (50.0 g, 347 mmol) in DMF (800 mL) at 0° C. wasadded NaH (60% dispersion in mineral oil, 15.3 g, 381 mmol). Thesuspension was stirred for 1 h at 0° C., and allyl bromide (29.3 mL, 347mmol) was then added to the reaction vessel using a syringe pump over 1h at 0° C. The reaction was warmed to rt overnight while stirring, andthe reaction mixture was then quenched with water (800 mL). The aqueouslayer was extracted with ether (5×400 mL), and the combined organicfractions were washed with water (3×500 mL) and brine (1×300 mL) andthen dried over MgSO₄. Chromatography of the crude product (8:1 to 2:1hexanes:EtOAc) yielded a clear oil (33.2 g, 52%, R_(f)=0.30 in 2:1hexanes/EtOAc). ¹H NMR (CDCl₃, 400 MHz): δ 1.48 (m, 2H), 1.62 (m, 4H),1.86 (m, 6H), 3.40 (t, 1H, J=8.8 Hz), 3.80 (t, 1H, J=9.0 Hz), 3.95 (d,2H, J=5.6 Hz), 5.15 (d, 1H, J=10.0 Hz), 5.26 (dd, 1H, J=1.2, 17.2 Hz),5.90 (m, 1H). ¹³C NMR (CDCl₃, 100 MHz): δ 20.6, 32.7, 36.3, 69.1, 71.6,78.6, 116.4, 135.3. FAB-HRMS: Calcd. for C₁₁H₂₁O₂ ⁺ [M+H]⁺: 185.1542.found: 185.1544.

Allylated cyclooctanone (8). To a stirring solution of alcohol 7 (33.2g, 180 mmol) in CH₂Cl₂ (650 mL) at rt was added pyridiniumchlorochromate (54.4 g, 252 mmol) over 2 h, 6.8 g every 15 min. After anadditional 30 min of stirring, the reaction mixture was concentrated ona rotary evaporator and the product was purified directly by columnchromatography (8:1 to 3:1 hexanes/EtOAc) to yield a clear oil (30.0 g,91%, R_(f)=0.30 in 5:1 hexanes/EtOAc). ¹H NMR (CDCl₃, 400 MHz): δ1.63-1.90 (m, 6H), 2.04 (m, 2H), 2.28 (m, 2H), 2.55 (m, 2H), 3.16 (tt,1H, J=2.8, 8.6 Hz), 3.89 (d, 1H, J=5.2 Hz), 5.13 (d, 2H, J=10.4 Hz),5.22 (dd, 1H, J=1.2, 17.2 Hz), 5.85 (ddd, 1H, J=5.6, 10.4, 22.2 Hz). ¹³CNMR (CDCl₃, 100 MHz): δ 22.9, 33.7, 42.3, 69.5, 77.6, 116.7, 135.0,216.5. FAB-HRMS: Calcd. for C₁₁H₁₉O₂ ⁺ [M+H]⁺: 183.1385. found:183.1380.

Silyl enol ether (9). To a stirring solution of Lithiumhexamethyldisilazide (LHMDS, 181 mL, 181 mmol, 1.00 M solution in THF)in THF (750 mL) at −78° C. was added a solution of ketone 8 (30.0 g, 165mmol) in THF (40 mL) over 1 h using a syringe pump. After an additional20 min of stirring at −78° C., chlorotriethylsilane (31.8 mL, 189 mmol)was added. The solution was stirred at −78° C. for 10 min, removed fromthe cold bath, warmed to rt with a water bath, and stirred for 1 h. Thereaction mixture was concentrated on a rotary evaporator and the crudeproduct was purified directly by column chromatography (100% hexanes to25:1 hexanes/EtOAc) to yield a clear oil (44.2 g, 91%, R_(f)=0.70 in 9:1hexanes/EtOAc). ¹H NMR (CDCl₃, 400 MHz): δ 0.67 (q, 6H, J=8.0 Hz), 0.98(t, 9H, J=8.0 Hz), 1.52 (m, 2H), 1.70-2.01 (m, 5H), 2.13 (m, 2H), 2.26(m, 1H), 3.40 (m, 1H), 3.95 (m, 2H), 4.76 (dd, 1H, J=7.2, 9.2 Hz), 5.14(dd, 1H, J=1.6, 10.4 Hz), 5.26 (dd, 1H, J=1.8, 15.4 Hz), 5.91 (ddd, 1H,J=5.4, 10.6, 22.6 Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 5.0, 6.7, 22.4, 24.5,31.8, 33.7, 36.2, 69.2, 79.9, 104.3, 116.3, 135.5, 152.7. Calcd. forC₁₇H₃₃O₂Si⁺ [M+H]⁺: 297.2250. found: 297.2246.

Monofluoroketones (10a and 10b). To a stirring solution of Selectfluor™(63.4 g, 179 mmol) in DMF (150 mL) at 0° C. was added a solution ofsilyl enol ether 9 (44.2 g, 149 mmol) DMF (180 mL) via an additionfunnel over 30 min. The reaction was allowed to slowly warm to rt whilestirring over 30 min, and then it was quenched with water (350 mL). Theaqueous layer was extracted with ether (4×350 mL), and the combinedorganic extracts were washed with water (3×300 mL), and brine (1×200mL). The crude product was purified by column chromatography (10:1 to5:1 hexanes/EtOAc) to yield two diastereomers, both clear oils (10a(cis), 19.5 g, 65%, R_(f)=0.40 in 9:1 hexanes/EtOAc and 10b (trans),9.10 g, 30%, R_(f)=0.20 in 9:1 hexanes/EtOAc). Relative stereochemistrywas assigned upon determination of the x-ray crystal structure of 11, adecomposition product of 10b (Supporting FIG. 6).

10a (trans): ¹H NMR (CDCl₃, 400 MHz): 1.59 (m, 1H), 1.69 (m, 2H), 1.90(m, 1H), 2.02 (m, 1H), 2.16-2.42 (m, 3H), 2.88 (m, 1H), 3.26 (m, 1H),3.91 (m, 2H), 4.92 (ddd, 1H, J=2.6, 6.4, 50.4 Hz), 5.16 (dd, 1H, J=1.6,10.4 Hz), 5.25 (dd, 1H, J=1.6, 17.2 Hz), 5.91 (ddd, 1H, J=5.2, 10.4,22.4 Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 20.1 (d, J=5 Hz), 27.2 (d, J=3Hz), 30.7 (d, J=21 Hz), 33.4, 40.2, 69.4, 77.5, 95.8 (d, J=185 Hz),116.8, 134.8, 213.5 (d, J=24 Hz). ¹⁹F NMR (CDCl₃, 376 MHz): δ −189.1(app t, J=43 Hz) FAB-HRMS: Calcd. for C₁₁H₁₈FO₂ ⁺ [M+H]⁺: 201.1291.found: 201.1291.

10b (trans): ¹H NMR (CDCl₃, 400 MHz): 1.62-1.91 (m, 4H), 1.93-2.11 (m,3H), 2.27-2.55 (m, 3H), 3.35 (m, 1H), 3.92 (m, 2H), 4.99 (ddd, 1H,J=3.4, 6.8, 48.0 Hz), 5.15 (dd, 1H, J=1.6, 10.4 Hz), 5.24 (dd, 1H,J=1.8, 17.2 Hz), 5.91 (ddd, 1H, J=5.6, 10.8, 22.8 Hz). ¹³C NMR (CDCl₃,100 MHz): δ 22.2, 26.6 (d, J=22 Hz), 27.0 (d, J=4 Hz), 31.7, 38.5, 69.5,76.0, 93.9 (d, J=184 Hz), 116.8, 134.9, 210.3 (d, J=17 Hz). ¹⁹F NMR(CDCl₃, 376 MHz): δ −188.0 (app t, J=41 Hz) FAB-HRMS: Calcd. forC11H₁₈FO₂ ⁺ [M+H]⁺: 201.1291. found: 201.1286.

Monofluoroketone (10b, trans). To a stirring solution of compound 10a(26.5 g, 132 mmol) in THF (300 mL) at 0° C. was added KHMDS (2.64 mL,1.32 mmol, 0.500 M solution in toluene). After 1 h, the reaction mixturewas concentrated on a rotary evaporator and the crude product waspurified by column chromatography (10:1 to 5:1 hexanes:EtOAc) to yieldtwo diastereomers, both clear oils (10a, 14.4 g, 54%, and 10b, 11.8 g,45%).

Fluorinated silyl enol ether (12). To a stirring solution of Potassiumhexamethyldisilazide (KHMDS, 225 mL, 113 mmol, 0.500 M solution intoluene) in THF (800 mL) at −78° C. was added a solution of ketone 10b(18.8 g, 93.9 mmol) in THF (40 mL) dropwise, using a syringe pump, over2 h. After an additional 30 min of stirring at −78° C.,chlorotriethylsilane (20.5 mL, 122 mmol) was added. The solution wasstirred at −78° C. for 30 min, removed from the cold bath, and stirredfor 1 h at rt. The reaction mixture was concentrated on a rotaryevaporator and the crude product was purified directly by columnchromatography (100% hexanes to 25:1 hexanes/EtOAc) to yield a clear oil(28.7 g, 97% as a 5:1 mixture of desired:undesired regioisomers,R_(f)=0.70 in 9:1 hexanes/EtOAc). ¹H NMR (CDCl₃, 400 MHz): δ 0.67 (q,6H, J=8.0 Hz), 0.97 (t, 9H, J=8.0 Hz), 1.50-1.91 (m, 5H), 2.02-2.13 (m,2H), 2.14-2.30 (m, 2H), 2.27-2.55 (m, 1H), 3.43 (m, 1H), 3.96 (m, 2H),5.15 (dd, 1H, J=1.6, 10.4 Hz), 5.27 (dd, 1H, J=1.6, 17.2 Hz), 5.91 (ddd,1H, J=5.2, 10.4, 22.4 Hz). ¹³C NMR (CDCl₃, 100 MHz): δ 5.2, 6.7, 24.5(d, J=3 Hz), 24.5 (d, J=27 Hz), 31.2, 32.9, 33.5 (d, J=2 Hz), 69.3,79.0, 104.3, 116.4, 128.6 (d, J=81 Hz), 135.2, 144.2 (d, J=239 Hz). ¹⁹FNMR (CDCl₃, 376 MHz): δ −129.5 (dd, J=21, 28 Hz).

Allylated difluoroketone (13). To a stirring solution of Selectfluor™(35.8 g, 101 mmol) in DMF (100 mL) was slowly added a solution of silylenol ether 12 (24.4 g, 77.7 mmol) in DMF (180 mL) over 30 min using anaddition funnel, at 0° C. The reaction mixture was allowed to warm tort, and, after 2 h of additional stirring, the reaction mixture wasquenched with water (100 mL). The aqueous layer was extracted with ether(4×200 mL), and the combined organic extracts were washed with water(3×200 mL) and brine (1×100 mL) and then dried over MgSO₄. The crudeproduct was purified by column chromatography (11:1 to 6:1hexanes/EtOAc) to yield a clear oil (12.5 g, 74%, R_(f)=0.60 in 4:1hexanes/EtOAc). ¹H NMR (CDCl₃, 300 MHz): 1.65-1.93 (m, 5H), 2.04-2.20(m, 2H), 2.27-2.47 (m, 1H), 2.48-2.61 (m, 1H), 2.75-2.85 (m, 1H), 3.35(m, 1H), 3.92 (dt, 2H, J=1.4, 5.7 Hz), 5.17 (dd, 1H, J=1.4, 10.4 Hz),5.25 (dd, 1H, J=1.5, 17.4 Hz), 5.91 (ddd, 1H, J=5.6, 10.8, 22.8 Hz). ¹³CNMR (CDCl₃, 100 MHz): δ 21.0, 25.9 (t, J=6 Hz), 31.0 (t, J=25 Hz), 31.4,38.0, 69.4, 75.9, 116.9, 118.5 (t, J=250 Hz), 134.7, 203.4 (t, J=28 Hz).¹⁹F NMR (CDCl₃, 376 MHz): δ −105.0 (app q, J=244 Hz) FAB-HRMS: Calcd.for C₁₁H₁₇F₂O₂ ⁺ [M+H]⁺: 219.1197. found: 219.1223.

Difluoroketone carboxylic acid (14). To a stirring solution of alkene 13(4.90 g, 22.5 mmol) in CCl₄ (45 mL), CH₃CN (45 mL), and H₂O (68 mL) at0° C. in a three-necked flask equipped with an overhead stirrer wasadded NaIO₄ (19.3 g, 90.0 mmol) and RuCl₃.H₂O (117 mg, 0.563 mmol).After 10 min of vigorous stirring at 0° C., the reaction was allowed towarm to rt. After an additional 2.5 h of vigorous stirring of thesuspension at rt, the reaction mixture was concentrated on a rotaryevaporator and diluted with 1 N HCl (200 mL) and brine (200 mL). Theaqueous layer was extracted with CH₂Cl₂ (7×200 mL) and dried extensivelyover MgSO₄. The crude product was purified by column chromatography (4:1to 1:1 hexanes/EtOAc, with 1% acetic acid) to yield a clear oil whichturned to a white solid upon storage at −20° C. overnight (5.10 g, 96%,R_(f)=0.30 in 1:1 hexanes/EtOAc with 1% acetic acid). ¹H NMR (CD₃CN, 400MHz): 1.62-1.97 (m, 5H), 1.98-2.20 (m, 2H), 2.27-2.42 (m, 1H), 2.53 (m,1H), 2.72 (m, 1H), 3.35 (tt, 1H, J=3.6, 7.6 Hz), 3.99 (s, 2H). ¹³C NMR(CD₃CN, 100 MHz): δ 22.1, 26.4 (t, J=6 Hz), 31.0 (t, J=25 Hz), 31.5,38.4, 66.2, 78.6, 119.8 (t, J=248 Hz), 172.2, 204.2 (t, J=28 Hz). ¹⁹FNMR (CD₃CN, 376 MHz): 6-105.9 (app t, J=15 Hz) FAB-HRMS: Calcd. forC₁₀H₁₅F₂O₄ ⁺ [M+H]⁺: 237.0938. found: 237.0934.

Vinyl triflate (15). To a solution of KHMDS (106 mL, 52.9 mmol, 0.500 Msolution in toluene) in THF (700 mL) was added a solution of ketone 14(6.10 g, 25.8 mmol) in THF (20 mL) at −78° C. dropwise over 20 min.After an additional 1 h of stirring at −78° C., a solution ofN-phenyl-bis(trifluoromethylsulfonamide) (Tf₂NPh, 18.9 g, 52.9 mmol) inTHF (40 mL) was added and the reaction was allowed to warm to rt. After30 min, the reaction mixture was concentrated on a rotary evaporator andthe crude product was diluted with CH₂Cl₂ (200 mL), 1 N HCl (200 mL) andbrine (200 mL). The aqueous layer was extracted with CH₂Cl₂ (5×150 mL)and dried extensively with MgSO₄. The crude product was purified bycolumn chromatography (4:1 to 1:1 hexanes/EtOAc with 1% acetic acid) toyield the desired product (4.50 g, 47%, R_(f)=0.35 in 1:1 hexanes/EtOAcwith 1% acetic acid) as a colorless oil. ¹H NMR (CD₃CN, 400 MHz): δ 1.74(m, 1H), 1.92 (m, 3H), 2.21-2.43 (m, 2H), 2.58-2.74 (m, 2H), 3.59 (tt,1H, J=4.0, 7.8 Hz), 4.04 (d, 2H, J=1.6 Hz), 6.35 (td, 1H, J=2.8, 9.6Hz). ¹³C NMR (CD₃CN, 100 MHz): δ 19.8, 26.4 (t, J=2 Hz), 31.7, 32.1 (t,J=25 Hz), 66.4, 77.6, 119.8 (t, J=237 Hz), 130.6 (t, J=5 Hz), 143.0 (t,J=29 Hz), 172.2. ¹⁹F NMR (CD₃CN, 376 MHz): δ −74.4 (s, 3F), −89.2 (dd,2F, J=30, 273 Hz), −92.6 (dd, 2F, J=30, 273 Hz). FAB-HRMS: Calcd. forC₁₁H₁₃F₅O₆SLi⁺ [M+H]⁺: 375.0513. found: 375.0509.

DIFO (1). To a solution of diisopropylamine (10.1 mL, 71.3 mmol) in THF(86 mL) at −78° C. was added nBuLi (23.8 mL, 59.4 mmol, from a 2.5 Msolution in hexanes) dropwise and stirred at −78° C. for 45 min. In aseparate flask, a solution of vinyl triflate 15 (7.31 g, 19.8 mmol) inTHF (400 mL) was prepared and kept at −15° C. using a MeOH/ice bath. LDAwas added to this solution dropwise, via syringe pump, at the rate of1.0 equiv (˜40 mL of the solution prepared above) per 30 min, withvigorous stirring, until 2.5 equiv of LDA was added. Note: the colour ofthe solution turned from clear to yellow to amber. The reaction mixturewas quenched by the addition of 10 mL of saturated ammonium chloridesolution and then the solvent was removed by rotary evaporation. To theresidue was added CH₂Cl₂ (100 mL), 1 N HCl (50 mL), and brine (50 mL).The aqueous layer was extracted with CH₂Cl₂ (5×40 mL) and washedextensively with MgSO₄. The crude product was purified by columnchromatography (4:1 to 1:1 hexanes/EtOAc with 1% acetic acid) to yieldthe desired product (466 mg, 11%, R_(f)=0.35 in 1:1 hexanes/EtOAc with1% acetic acid) as a colorless oil. ¹H NMR (CD₃CN, 400 MHz): δ 1.94 (m,1H), 2.07 (m, 1H), 2.11-2.33 (m, 3H), 2.41 (m, 1H), 2.52 (m, 2H), 3.55(app t, J=7.2 Hz, 1H), 4.05 (s, 2H). ¹³C NMR (CD₃CN, 100 MHz): δ 17.6,34.4 (d, J=6 Hz), 39.2, 43.2 (t, J=28 Hz), 66.5, 84.4, 85.2 (dd, J=48,43 Hz), 113.9 (t, J=11 Hz), 120.5 (t, J=233 Hz), 172.2. ¹⁹F NMR (CD₃CN,376 MHz): δ −86.6 (dddt, J=259.0, 26.0, 12.8, 6.0 Hz, IF), −88.6 (dm,J=260.9 Hz, 1F). FAB-HRMS: Calcd. for C₁₀H₁₂F₂O₃Li⁺ [M+H]⁺: 225.0915.found: 225.0912.

General procedure for synthesis of conjugates of DIFO (5a-f). Thepentafluorophenyl ester of DIFO (16) was prepared as follows and usedimmediately. DIFO (1, 1.0 equiv) was dissolved in CH₂Cl₂ (finalconcentration of 0.1-0.2 M) and N,N-diisopropylethylamine (2.0 equiv)was added. The solution was cooled to 0° C. andpentafluorophenyltrifluoroacetate (1.05 equiv) was added dropwise. After1 h, the solvent and residual pentafluorophenol was removed on a rotaryevaporator. The crude product was either used immediately or quicklypurified by column chromatography (R_(f)=0.3 in 9:1 hexanes:ethylacetate) and then used immediately. ¹H NMR (CD₃CN, 400 MHz): δ 1.99 (m,1H), 2.09 (m, 1H), 2.15-2.35 (m, 3H), 2.43 (m, 1H), 2.54 (m, 2H), 3.63(app t, J=7.2 Hz, 1H), 4.53 (s, 2H). ¹⁹F NMR (CD₃CN, 376 MHz): δ −86.6(dddt, J=259.0, 26.0, 12.8, 6.0 Hz, 1F), −88.6 (dm, J=260.9 Hz, 1F),−153.6 (d, J=19 Hz, 2F), −159.2 (t, 21 Hz, 1F), −163.6 (dd, J=17, 4 Hz,2F).

Synthesis of Alexa Fluor derivatives (5a, DIFO-488; 5d, DIFO-568; 5e,DIFO-647; 6a, Alk-488). To a solution of the appropriate Alexa Fluorcadaverine (1.0 equiv) in DMF (final concentration of 0.2 M) was added asolution of the pentafluorophenyl ester of DIFO (16) or 4-pentynoic acid(2.0 equiv) and then N,N-diisopropylethylamine (5.0 equiv). The solutionwas stirred at rt overnight in the dark, and then the solvent wasremoved on a rotary evaporator. The residue was dissolved in water or9:1 water:acetonitrile, purified by reversed phase HPLC using water andacetonitrile, and lyophilized to a fine powder.

DIFO-488, FT-ICR-MS: Calcd. for C₃₆H₃₆F₂N₄O₁₂S₂ ⁺ [M⁺]: 818.1739. found:818.1725.

DIFO-568, FT-ICR-MS: Calcd. for C₄₈H₅₂F₂N₄O₁₂S₂ ⁺ [M⁺]: 978.2986. found:978.2971.

DIFO-647, FT-ICR-MS: Found: 1142.3509.

Alk-488, FT-ICR-MS: Calcd. for C₃₁H₃₀N₄O₁₁S₂ ⁺ [M⁺]: 698.1352. found:698.1318.

DIFO-biotin (5b). To a solution of biotin-PEG-amine (2) (15 mg, 0.034mmol) in DMF (0.5 mL) was added a solution of pentafluorophenyl ester 16(13 mg, 0.034 mmol) in DMF (1.0 mL) and then N,N-diisopropylethylamine(9.0 μL, 0.051 mmol). The solution was stirred overnight at rt, the DMFwas removed on a rotary evaporator, and the residue was purified bysilica gel chromatography (100% CH₂Cl₂ to 9:1 CH₂Cl₂:MeOH) to yield 19mg (87%) of a clear oil (R_(f) in 9:1 CH₂Cl₂:MeOH=0.40). ¹H NMR (MeOD,500 MHz): δ 1.43 (m, 2H), 1.55-1.70 (m, 3H), 1.71-1.83 (m, 5H), 1.95 (m,1H), 2.07-2.35 (m, 4H), 2.18 (t, J=8.0 Hz, 2H), 2.43 (m, 1H), 2.52 (m,2H), 2.70 (d, J=12.5 Hz, 1H), 2.92 (dd, J=12.5, 5.0 Hz, 1H), 3.20 (m,1H), 3.26 (t, J=6.8 Hz, 2H), 3.34 (t, J=6.8 Hz, 2H), 3.53 (m, 5H), 3.59(m, 4H), 3.64 (m, 4H), 3.95 (d, J=3.5 Hz, 2H), 4.30 (dd, J=7.5, 4.5 Hz,1H), 4.49 (dd, J=7.5, 5.0 Hz, 1H). ¹³C NMR (MeOD, 125 MHz): δ 18.2,27.4, 30.0, 30.3, 30.9, 35.3 (d, J=6 Hz), 37.4, 38.3 (d, J=4 Hz), 40.3,41.6, 44.2 (t, J=28 Hz), 57.5, 62.1, 63.9, 69.4, 70.4, 70.8, 71.8, 72.0,72.1, 85.2, 86.4 (dd, J=48, 42 Hz), 113.9 (t, J=11 Hz), 120.5 (t, J=234Hz), 166.6, 172.9, 176.5. ¹⁹F NMR (MeOD, 376 MHz): δ −88.0 (dddt,J=266.5, 26.3, 12.4, 6.0 Hz, IF), −90.0 (dm, J=259.4 Hz, IF). FAB-HRMS:Calcd. for C₃₀H₄₈F₂N₄O₇SLi⁺ [M+H]⁺: 653.3372. found: 653.3379.

Maleimide-amine (17). A dry 500 mL round-bottom flask was charged withmaleimide (3.12 g, 32.2 mmol) and triphenylphosphine (8.29 g, 31.6mmol), and then THF (150 mL). N-(tert-Butoxycarbonyl)ethanolamine (5.00mL, 29.3 mmol) and diisopropylazidodicarboxylate (6.80 mL, 35.1 mmol)were added in succession. The flask was stirred under a nitrogenatmosphere overnight, the reaction mixture was concentrated on a rotaryevaporator, and the crude product was filtered through a plug of silicagel using a 2:1 mixture of hexanes:ethyl acetate as the eluent. Thecrude product was dissolved in 100 mL of a 60:35:5 mixture ofCH₂Cl₂:trifluoroacetic acid:water and stirred at rt for 2 h. Thereaction mixture was diluted with CH₂Cl₂ (50 mL) and water (50 mL),transferred to a separatory funnel, and the organic layer was extractedwith water (3×25 mL). The combined aqueous layers were washed withCH₂Cl₂ (3×50 mL) and concentrated on a rotary evaporator to yield thedesired product (7.51 g, 96%) as a yellow oil. ¹H NMR (DMSO-d₆, 400MHz): δ 1.76 (app quintet, 2H, J=7.2 Hz), 2.76 (m, 2H), 3.45 (t, 2H,J=6.8 Hz), 7.02 (s, 2H), 7.79 (br s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz): δ26.7, 34.6, 37.0, 115.6 (q, J=290 Hz), 134.7, 158.9 (q, J=36 Hz), 171.3.¹⁹F NMR (DMSO-d₆, 376 MHz): δ −73.9.

DIFO-maleimide (5f). To a solution of maleimide-amine 17 (74 mg, 0.27mmol) in CH₂Cl₂ (1.5 mL) was added N,N-diisopropylethylamine (160 μL,0.91 mmol). The solution was cooled to 0° C. and then a solution ofpentafluorophenyl ester 16 (97 mg, 0.23 mmol) in CH₂Cl₂ (1 mL) was addeddropwise. The reaction mixture was stirred for 2 h, the solvent wasremoved on a rotary evaporator, and the residue was purified by silicagel chromatography (2:1 to 1:3 hexanes:ethyl acetate) to yield 62 mg(76%) of a clear oil (R_(f)=0.20 in 1:1 hexanes:ethyl acetate). ¹H NMR(CDCl₃, 500 MHz): δ 1.81 (app q, J=6.5 Hz, 2H), 2.05-2.31 (m, 5H), 2.48(m, 2H), 2.60 (m, 1H), 3.26 (t, J=6.5 Hz, 2H), 3.55 (t, J=6.8 Hz, 1H),3.60 (t, J=6.5 Hz, 2H), 3.95 (dd, J=20.5, 15.0 Hz, 2H), 6.74 (s, 2H),7.12 (br s, 1H). ¹⁹F NMR (CD₃CN, 376 MHz): 6-86.6 (dddt, J=266.5, 26.3,12.4, 6.0 Hz, 1F), −88.5 (dm, J=259.4 Hz, 1F).

DIFO-FLAG (5c). Cysteine-modified FLAG peptide (FLAG-C,H₂N-DYKDDDDKC-CO₂H) was synthesized using established automatedprotocols on a Perkin-Elmer ABI 431 A peptide synthesizer usingfluorenylmethoxycarbonyl (Fmoc)-Cys(Trt)-Wang resin (Novabiochem). Thepeptide was cleaved using a solution of trifluoroaceticacid:triisopropylsilane:water (95:2.5:2.5), precipitated with ether, andthe crude product was dried and used without further purification. To asolution of FLAG-C (214 mg, 0.192 mmol) in 1 mL of water was added asolution of DIFO-maleimide (5f, 62 mg, 0.175 mmol) in 1 mL of DMF at 0°C. The reaction mixture was allowed to warm to rt and stir overnight.The solvents were removed on a rotary evaporator and the residue waspurified by reversed phase HPLC (5% to 40% acetonitrile in water over 60min, product eluting at 30-35 min) and lyophilized to yield 125 mg (49%)of a white solid. MALDI-TOF: Calcd. [M+H]⁺: 1470.5540. found: 1470.5526.

Kinetic Evaluation of the [3+2] Cycloaddition of DIFO and Benzyl Azide

Stock solutions of DIFO (1, 20 mM) and benzyl azide (200 mM) were madein CD₃CN. An NMR tube was charged with 450 μL of the solution of 1, and,immediately before lowering into the NMR magnet, 50 μL of the benzylazide solution, and the reaction was monitored over time using ¹H NMRspectroscopy. The kinetic data were derived by monitoring the change inintegration of resonances corresponding to the benzylic protons inbenzyl azide (δ ˜4.4 ppm) compared to the corresponding resonances ofthe triazole products (δ ˜5.5 to 5.7 ppm). The second-order rateconstant for the reaction was determined by plotting 1/[benzyl azide]versus time, followed by subsequent analysis by linear regression Thesecond-order rate constant (k, M⁻¹ s⁻¹) corresponds to the determinedslope.

In vivo labeling of glycoproteins with azido sugars and DIFO-FLAG. Forin vivo metabolic labeling experiments, B6D2F1 mice were administereddaily doses of Ac₄ManNAz (300 mg/kg in ˜150 μL of 70% DMSO, from a stocksolution of 50 mg/mL) intraperitoneally (i.p.) for 7 d as previouslydescribed (Prescher et al. Nature 2004). Sixteen to 24 h after the finalazidosugar bolus, mice were injected i.p. with DIFO-FLAG (5c, 0.16 mmolkg⁻¹ in 70% DMSO) or vehicle (70% DMSO). After 3 h, the mice weresacrificed and organs were harvested.

To determine the extent of ligation of azides with DIFO-FLAG in vivo,splenocytes from B6D2F1 mice treated with the appropriate combination ofAc₄ManNAz, DIFO-FLAG or vehicle (70% DMSO), as described above, wereisolated and probed for the presence of cell-surface Flag epitopes usinga flow cytometry assay. Briefly, splenocytes from one spleen weresuspended in RPMI medium 1640 and distributed among wells of a 96-wellV-bottom tissue culture plate (three wells per treatment, ˜5×10⁵cells/well). The cells were pelleted, rinsed with labeling buffer (PBS,pH 7.4 containing 1% FBS), and incubated with a FITC-conjugatedanti-FLAG antibody (1:900 dilution) in labeling buffer for 30 min onice. All cells were analyzed by flow cytometry.

The data are shown in FIG. 9. FIG. 9 depicts in vivo reaction ofDIFO-FLAG with metabolically labeled azido glycans. MFI, meanfluorescence intensity. B6D2F1 mice were injected once daily for 7 dayswith 300 mg/kg Ac₄ManNAz in 70% DMSO (+) or vehicle (−). On the eighthday, the mice were injected with a single bolus of DIFO-FLAG in PBS(0.16 mmol/kg) or vehicle and sacrificed 3 hours post-injection.Splenocytes were harvested, stained with FITC-labeled anti-FLAGantibody, and analyzed by flow cytometry. Shown is the mean fluorescenceintensity (MFI, in arbitrary units). Each diamond represents data from asingle mouse.

The data presented in FIG. 9 demonstrate that DIFO-FLAG can selectivelylabel azide-containing glycans inside a living mouse, specifically inthe cells of the spleen.

Comparative Labeling of Azido Proteins by Cu-Free and Cu-Catalyzed ClickChemistries. Recombinant murine dihydrofolate reductase was expressedwith (azido-DHFR) or without (DHFR) replacement of its methionineresidues by azidohomoalanine as previously described (Kiick et al. Proc.Natl. Acad. Sci. U.S.A. 2002) and stock solutions were normalized to 0.1mg/mL in PBS in 1% sodium dodecylsulfate (SDS). Stock solutions weremade of DIFO-488 and alk-488 in PBS (1 mM), CuSO₄ in water (20 mM),tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in water (20 mM), andtris-triazolyl ligand (TBTA, 1.7 mM in 4:1 t-butanol:dimethylsulfoxide(DMSO)). Reactions were performed in 10 μL volumes at finalconcentrations of the following reagents: 1 μg/mL azido-DHFR or DHFR, 25μM of DIFO-488 or alk-488, 0.04% SDS, 1×PBS, pH 7.4. In addition, inaccordance with conditions optimized by Speers and Cravatt ((2004) Chem.Biol. 11:535-46), the Cu-catalyzed reactions with alk-488 also containedCuSO₄ (1 mM), TCEP (1 mM), and TBTA (100 μM), such that the finalconcentrations of t-butanol and DMSO were 4.8% and 1.2%, respectively)(28). The order of addition was as follows: (a) Cu-free click chemistry,PBS, azido-DHFR or DHFR, and DIFO-488; (b) Cu-catalyzed click chemistry,PBS, azido-DHFR or DHFR, alk-488, TCEP, ligand, and CuSO₄. Reactionswere started by addition of the final reagent, briefly vortexed, andallowed to sit in the dark at rt for 0-60 min. Reactions were quenchedby the sequential addition of 1 μL of a solution of 2-azidoethanol inwater (500 mM) and 11 μL of a solution of urea in water (8 M) for finalconcentrations of 23 mM and 4 M, respectively, followed by briefvortexing and an additional hour in the dark at rt. (For t=0 min, the2-azidoethanol and urea were added to the reaction mixture after theprotein was added, followed by brief vortexing, and before the otherreagents.) SDS protein loading buffer containing β-mercaptoethanol (4×,7.3 μL) was added, the samples were heated at 100° C. for 10 min, andloaded onto 12% Bis-Tris Criterion polyacrylamide gel (Bio-Rad). Afterelectrophoresis, gels were rinsed in a destain solution (5:4:1water:methanol:acetic acid) overnight (in the dark) and fluorescenceintensities were measured using a Typhoon 9410 (GE Healthcare). Equalprotein loading was confirmed using silver stain (Bio-Rad).

The data are presented in FIG. 10. FIG. 10 depicts time-dependentlabeling of an isolated azidoprotein by Cu-catalyzed (lower panedl) orCu-free (DIFO; upper panel) click chemistry, using Alexa Fluor 488derivatives. Recombinant murine dihydrofolate reductase was expressed inE. coli with media supplemented either with methionine (−) orazidohomoalanine

(all other lanes), and purified (see Kiick et al. (2002) Proc. Natl.Acad. Sci. U.S.A 99:19). The purified protein was reacted for varioustimes (0-60 min) with an Alexa Fluor 488 cadaverine derivative of thedifluorinated cyclooctyne DIFO (“Cu-free click chemistry”) or pentynoicacid (“Cu-catalyzed chemistry”) for the indicated times. In the case ofCu-catalyzed click chemistry, the reaction was supplemented with 1 mMCuSO₄, 1 mM TCEP, and 100 μM of the tris-triazolyl ligand TBTA accordingto the conditions of Speers and Cravatt (2004) Chem. Biol. 11:535-46.The reactions were performed in PBS, quenched by the addition of 8 Murea and 10 mM azidoethanol, and analyzed by SDS-PAGE and in-gelfluorescence measurement.

The data presented in FIG. 10 demonstrate that DIFO can selectivelylabel an azide-containing isolated protein with similar reactionkinetics to copper-catalyzed click chemistry using terminal alkynes,Cu(I) salts, TCEP, and the tris-trazolyl ligand TBTA.

Cell surface labeling of azido glycans on Jurkat cells with biotinylatedconjugates. Jurkat (human T-cell lymphoma) and Chinese hamster ovary(CHO) cells were maintained in a 5% CO₂, water-saturated atmosphere andgrown in RPMI-1640 (Jurkat) or F12 (CHO) media supplemented with 10%FCS, penicillin (100 units/mL), and streptomycin (0.1 mg/mL). Celldensities were maintained between 1×10⁵ and 1.6×10⁶ cells/mL.

Jurkat cells were incubated for 1-3 d in untreated media or mediacontaining 25 μM Ac₄ManNAz. The cells were then distributed into a96-well V-bottom tissue culture plate, pelleted (3500 rpm, 3 min), andwashed twice with 200 μL of labeling buffer (PBS, pH 7.4 containing 1%FCS). Cells were then incubated with DIFO-biotin or a similarbiotinylated analog of a nonfluorinated or a triaryl phosphine capableof Staudinger ligation in labeling buffer for 1 h at rt at variousconcentrations (10 nM to 100 μM) with dilutions made from a 2.5 mM stockin 7:3 PBS:DMF. After incubation, cells were pelleted, washed twice withlabeling buffer, and resuspended in the same buffer containingfluorescein isothiocyanate (FITC)-avidin (1:200 dilution of the Sigmastock). After a 15-min incubation on ice (in the dark), the cells werewashed once with 200 μL, incubated with FITC-avidin for an additional 15min on ice, washed twice with 200 μL of cold labeling buffer, and thendiluted to a volume of 400 μL for flow cytometry analysis.

The data are shown in FIGS. 11A and 11B. FIGS. 11A and 11B depict cellsurface labeling of azido glycans by various biotinylated derivatives ofcyclooctynes or phosphines. Jurkat cells were incubated with 0 or 25 μMAc₄ManNAz for 3 days. (A) The cells were then reacted with 100 μMphosphine-biotin (Vocadlo et al. Proc. Natl. Acad. Sci. U.S.A. 2003,100, 9116-9121), cyclooctyne-biotin, or DIFO-biotin for 1 h. (B) Thecells were reacted with various concentrations of DIFO-biotin for 1 h.In both cases, the cells were then stained with FITC-avidin, andanalyzed by flow cytometry. Shown is the mean fluorescence intensity(MFI, arbitrary units). Error bars represent the standard deviation oftriplicate samples.

The data presented in FIGS. 11A and 11B show that DIFO-biotin canselectively label azide-containing membrane-associated glycans in livecells; further, the sensitivity of azide detection is much higher usingDIFO than a nonfluorinated cyclooctyne or a phosphine capable ofStaudinger ligation.

Cell surface labeling of azido glycans on CHO cells and imaging byfluorescence microscopy. CHO cells were incubated for 2 d in mediacontaining 100 μM Ac₄ManNAz or Ac₄ManNAc in an eight-well LabTek IIchambered coverglass (Nunc). The media was gently aspirated and thecells were washed three times with 600 μL of complete media. The cellswere then treated with a solution of DIFO-488, DIFO-568, or DIFO-647,diluted from a 1 mM stock solution in PBS (pH 7.4), in media for varyingtimes (0-60 min) at varying concentrations (10-100 μM) and temperatures(4-37° C.). For long time-course labeling studies, the cells were washedwith 600 μL of media three times after the labeling reaction andreturned to media containing 100 μM Ac₄ManNAz or Ac₄ManNAc until thenext labeling reaction. Immediately prior to imaging, the cells weretreated with Hoechst 33342 dye to stain the nucleus (1:1000 dilution inmedia of a 1 mg/mL stock solution in DMSO) for 1 min at rt, washed twicewith 600 μL of media, and imaged. Optimized conditions for exclusivecell surface labeling: (a) 100 μM DIFO-488 for 1 min in media pre-warmedto 37° C., or (b) 100 μM DIFO-488 for 1 h in media at 4° C. Optimizedconditions for cell surface and Golgi labeling: 10 μM DIFO-488 for 1 hin media at 37° C. For experiments with intracellular organelle markersfor lysosomes (LysoTracker Red™, Invitrogen) and Golgi (BODIPY TRceramide, Invitrogen) instructions provided by the manufacturer wereutilized. Propidium iodide, used to stain for cell viability, wasapplied (1:3000 dilution in media of a 1 mg/mL stock solution in waterfor 3 min at rt) immediately prior to imaging, after application ofHoechst dye; the cells were then washed twice with 600 μL of media andimaged.

The data demonstrate that DIFO-Alexa Fluor conjugates can selectivelylabel azide-containing membrane-associated glycans on CHO cell surfaces,e.g., for the purposes of imaging the dynamics of these biomolecules inlive cells on the minute and hour timescale.

Example 6 Synthesis and Characterization of Difluorinated Cycloocytyne

The following schemes depict synthesis of DIFO2 and DIFO3, and synthesisof biotin conjugates of DIFO2 and DIFO3.

Synthesis of DIFO2 (7) and DIFO3 (13)

1. A solution of 1,3-cyclooctanedione (1.80 g, 12.8 mmol) in MeCN (90mL) was transferred to a flame-dried round bottom flask under an N₂atmosphere, and the system was cooled to 0° C. with stirring. Cs₂CO₃(8.58 g, 26.3 mmol) was added, followed 15 min later by Selectfluor™(10.9 g, 30.8 mmol), and the reaction mixture was stirred for anadditional 15 min. The system was then allowed to warm to rt and wasstirred for 6 h, concentrated under reduced pressure, and diluted with 1M HCl (150 mL). The resulting mixture was extracted with diethyl ether(120 mL, 4×), and the combined organic layers were washed with brine(200 mL), dried over MgSO₄, and filtered through a glass frit. Thesolution was then concentrated under reduced pressure and purified byflash chromatography (4:1 hexanes:EtOAc) to yield a white solid (1.57 g,69%). R_(f)=0.40 (4:1 hexanes:EtOAc); mp 42.7-43.7° C.; ¹H NMR (500 MHz,CDCl₃) δ 2.67 (m, 4H), 1.81 (m, 4H), 1.63 (m, 2H) ppm; ¹³C NMR (125 MHz,CDCl₃) δ 197.9 (t, J=25.1 Hz), 109.5, 38.7, 26.2, 24.7 ppm; ¹⁹F NMR (376MHz, CDCl₃) δ −118.35 (s, 2F) ppm; HRMS (EI⁺) calcd for C₈H₁₀O₂F₂176.0649 found 176.0646.

3. To a flame-dried round bottom flask were added difluorodiketone 1(998 mg, 5.67 mmol), phosphonium iodide 2 (5.57 g, 11.34 mmol), and THF(100 mL). A N₂ atmosphere was established, and the system was cooled to0° C. DBU (1.27 mL, 8.51 mmol) was added, and the reaction mixture wasstirred for 20 min at 0° C. and then brought to rt. The reaction wasstirred an additional 24 h, quenched with MeOH (10 mL), concentratedunder reduced pressure, and purified by flash chromatography (20-75%toluene:hexanes) to yield a white solid (1.38 g, 79%). R_(f)=0.55 (4:1hexanes:EtOAc); mp 58.8-61.1° C.; ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, 2H,J=8.4 Hz), 7.38 (d, 2H, J=8.2 Hz), 7.23 (s, 1H), 3.92 (s, 3H), 2.70 (t,2H, J=6.6 Hz), 2.52 (apt t, 2H, 6.2 Hz), 1.86 (m, 2H), 1.53 (m, 4H) ppm;¹³C NMR (125 MHz, CDCl₃) δ 202.1 (t, J=28.9 Hz), 166.8, 140.0, 134.6 (t,J=19.6 Hz), 131.2 (t, J=10.3 Hz), 130.0, 129.7, 129.0, 115.2 (t, J=253.4Hz), 52.4, 37.5, 27.3, 26.0, 25.7, 25.3 ppm; ¹⁹F NMR (376 MHz, CDCl₃) δ−111.09 (s, 2F) ppm; HRMS (FAB) calcd for C₁₇H₁₈O₃F₂ [M+H]⁺ 309.1302found 309.1302.

4. To a round bottom flask were added olefin 3 (1.11 g, 3.60 mmol) andMeOH (40 mL). The system was flushed with N₂ and a catalytic amount ofPd/C was added. The system was again flushed with N₂ followed by H₂, andthe reaction was stirred under a H₂ atmosphere for 1.5 h. The system wasthen flushed with N₂ and the reaction was diluted with CH₂Cl₂ (40 mL),filtered through Celite, and concentrated under reduced pressure. Thecrude product was purified by flash chromatography (33-75%toluene:hexanes) to yield a white solid (1.09 g, 97%). R_(f)=0.60 (4:1hexanes:EtOAc); mp 70.1-71.4° C.; ¹H NMR (400 MHz, CDCl₃) δ 7.99 (dm,2H, J=8.3 Hz), 7.27 (d, 2H, J=8.14 Hz), 3.92 (s, 3H), 3.31 (dd, 1H,J=13.6, 2.9 Hz), 2.82-2.73 (m, 1H), 2.66-2.48 (m, 2H), 2.45-2.28 (m,1H), 2.17-2.02 (m, 1H), 1.97-1.84 (m, 1H), 1.66-1.42 (m, 4H), 1.41-1.29(m, 1H), 1.29-1.11 (m, 1H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 205.7 (dd,J=30.4, 25.1 Hz), 167.1, 144.72, 130.0, 129.4, 128.6, 119.4 (dd,J=258.0, 250.7 Hz), 52.2, 46.4 (t, J=21.6), 39.1, 33.8 (t, J=4.8 Hz),27.2, 24.3 (d, J=6.9 Hz), 24.1 (d, J=3.4 Hz), 22.9 ppm; ¹⁹F NMR (376MHz, CDCl₃) δ −102.72 (d, 1F, J=245.5 Hz), −122.67 (d, 1F, J=251.7 Hz)ppm; HRMS (FAB) calcd for C₁₇H₂₀O₃F₂ [M+H]⁺ 311.1459 found 311.1467.

5. To a flame-dried round bottom flask under a N₂ atmosphere was addedTHF (80 mL), followed by KHMDS (8.28 mL, 4.14 mmol). The reactionmixture was cooled to −78° C. with stirring, and ketone 4 (1.07 g, 3.45mmol) in THF (35 mL) was added dropwise over 15 min. The reaction wasstirred for an additional 40 min, then Tf₂NPh (1.36 g, 3.80 mmol) in THF(35 mL) was added via syringe, and the system was then brought to rtwith stirring over 1.5 h. The mixture was then quenched with MeOH (3 mL)concentrated under reduced pressure, diluted with ether, filteredthrough a silica gel plug, and again concentrated. The crude mixture wasthen flash chromatographed twice (0-5% EtOAc in 4:1 hexanes:toluene with1% Et₃N, then 0-1% EtOAc in 2:1 hexanes:toluene) to yield a pale yellowoil (1.13, 74%). R_(f)=0.60 (4:1 hexanes:EtOAc); ¹H NMR (400 MHz, CD₃CN)δ 7.93 (d, 2H, J=8.2 Hz), 7.36 (d, 2H, J=8.2 Hz), 6.25 (t, 1H, J=9.4Hz), 3.85 (s, 3H), 3.24 (dd, 1H, J=13.5, 3.8 Hz), 2.88-2.70 (m, 1H),2.61 (dd, 1H, J=13.5, 10.2 Hz), 2.56-2.42 (m, 1H), 2.41-2.30 (m, 1H),1.68-1.52 (m, 3H), 1.51-1.44 (m, 2H), 1.43-1.34 (m, 1H) ppm; ¹³C NMR(125 MHz, CD₃CN) δ 167.6, 145.8, 143.6 (t, J=29.4 Hz), 130.5, 130.4,129.5, 129.1 (t, J=4.0 Hz), 120.5 (dd, J=246.2, 243.1 Hz), 119.5 (q,J=319.1 Hz), 52.7, 46.8 (apt t, J=22.0 Hz), 35.2 (apt d, J=5.4 Hz),27.0, 26.2, 23.0, 21.8 ppm; ¹⁹F NMR (376 MHz, CD₃CN) δ −74.45 (s, 3F),−93.93 (d, 1F, J=272.2 Hz), −105.18 (d, 1F, J=266.9 Hz) ppm; HRMS (FAB)calcd for C₁₈H₁₉O₅F₅S [M+H]⁺ 443.0952 found 443.0960.

6. To a flame-dried round bottom flask under a N₂ atmosphere was addedvinyl triflate 5 (986 mg, 2.23 mmol) in THF (50 mL). The mixture wascooled to −20° C. with stirring. In a separate flame-dried round bottomflask, a 0.198 M solution of LDA was made by adding n-butyllithium (2.68mL, 6.69 mmol) to a solution of diisopropylamine (1.13 mL, 8.03 mmol) inTHF (30.0 mL) stirring under a N₂ atmosphere at −78° C. A portion of theLDA solution (13.5 mL, 2.67 mmol) was added dropwise to the firstmixture over 1 h. The reaction mixture was then brought to rt over 30min, quenched with MeOH (5 mL), concentrated under reduced pressure, andpurified by flash chromatography (0-3% EtOAc in 2:1 hexanes:toluene) toyield a white solid (529 mg, 81%). R_(f)=0.50 (1:2 hexane:toluene); mp78.5-82.7° C.; ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, 2H, J=8.1 Hz), 7.27(d, 2H, J=7.9 Hz), 3.92 (s, 3H), 3.16 (apt d, 1H, J=11.2 Hz), 2.60-2.43(m, 2H), 2.41-2.24 (m, 2H), 2.10-1.88 (m, 2H), 1.87-1.68 (m, 2H),1.62-1.44 (m, 1H), 1.21-1.08 (m, 1H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ167.2, 145.5, 130.0, 129.4, 128.4, 119.5 (t, J=238.6 Hz), 109.9 (t,J=11.1 Hz), 85.1 (dd, J=47.2, 41.6 Hz), 58.2 (t, J=24.3 Hz), 52.2, 34.5(d, J=4.7 Hz), 32.6, 30.8 (d, J=4.4 Hz), 28.0, 20.4 ppm; ¹⁹F NMR (376MHz, CDCl₃) δ −94.32 (d, 1F, J=260.2 Hz), −101.36 (dm, 1F, J=259.8 Hz)ppm; HRMS (FAB) calcd for C₁₇H₁₈O₂F₂ [M+H]⁺ 293.1353 found 293.1357.

7. To a round bottom flask fitted with a reflux condenser were addeddifluorocyclooctyne methyl ester 6 (442 mg, 1.51 mmol), LiOH (723 mg,30.2 mmol), water (1.75 mL), and dioxane (7 mL). The reaction was heatedto 55° C. and stirred for 7 h. The mixture was then cooled to rt,diluted with 1 M HCl (10 mL), and extracted with CH₂Cl₂ (20 mL, 4×). Thecombined organic layers were then washed (1:1 1 M HCl:brine, 40 mL),dried over Na₂SO₄, filtered through a glass frit, and concentrated underreduced pressure. The crude product was then purified by flashchromatography (9:1 hexanes:EtOAc with 1% AcOH) to yield a white solid(360 mg, 86%). R_(f)=0.45 (4:1 hexanes:EtOAc with 1% AcOH); mp 182.0(dec); ¹H NMR (400 MHz, CD₃CN) δ 10.10-8.80 (br s, 1H), 7.94 (d, 2H,J=8.2 Hz), 7.35 (d, 2H, J=8.1), 3.10 (d, 1H, J=10.9 Hz), 2.70-2.50 (m,2H), 2.43-2.24 (m, 2H), 2.04-1.84 (m, 2H) 1.83-1.67 (m, 2H), 1.55-1.41(m, 1H), 1.22-1.11 (m, 1H) ppm; ¹³C NMR (125 MHz, d₆-acetone) δ 167.6,146.2, 130.8, 130.2, 129.6, 120.2 (dd, J=239.1, 235.4 Hz), 111.6 (apt t,J=11.3 Hz), 85.5 (dd, J=46.7, 41.9 Hz), 58.6 (t, J=24.2 Hz), 34.9 (d,J=4.9 Hz), 33.3 (d, J=2.1 Hz), 31.6 (d, J=4.7 Hz), 28.4, 20.5 ppm; ¹⁹FNMR (376 MHz, CD₃CN) δ −93.81 (d, 1F, J=258.7 Hz), −101.32 (ddt, 1F,J=258.8, 20.3, 7.0 Hz) ppm; HRMS (FAB) calcd for C₁₆H₁₆O₂F₂ [M+H]⁺279.1197 found 279.1190.

8. To a flame-dried round bottom flask were added DCC (3.66 g, 17.7mmol), DMAP (98.6 mg, 0.807 mmol), 3-methyl-3-oxetanemethanol (1.59 mL,16.1 mmol), and CH₂Cl₂ (20 mL). A N₂ atmosphere was established, and themixture was cooled to 0° C. with stirring. Iodoacetic acid (3.00 g, 16.1mmol) was then added in CH₂Cl₂ (30 mL) via syringe. The reaction mixturewas stirred for 1 h, and then was brought to rt over 1.5 h, quenchedwith acetic acid (1 mL), and stirred an additional 30 min. The mixturewas then diluted with CH₂Cl₂ and filtered through Celite to remove DCU.The filtrate (300 mL total) was then washed with water (200 mL),saturated NaHCO₃ (200 mL, 2×) to remove residual acids, and brine (200mL). The organic layer was then dried over MgSO₄, filtered through aglass frit, and concentrated under reduced pressure. At this pointresidual DCU precipitated from the solution, so it was diluted withCH₂Cl₂ and again filtered through Celite. The filtrate was againconcentrated to yield a yellow oil. This material was then transferredto a new round bottom flask and dissolved in THF (100 mL). Triphenylphosphine (4.65 g, 17.7 mmol) was then added, and the reaction wasstirred under N₂ at rt for 40 h. The reaction mixture was then dilutedwith diethyl ether (100 mL) and filtered through a glass frit to isolatethe precipitated product. Residual solvent was removed under reducedpressure to yield a white solid (7.93 g, 92%). Mp 152.8-154.6° C.; ¹HNMR (400 MHz, CDCl₃) δ 7.93-7.84 (m, 6H), 7.84-7.78 (m, 3H), 7.73-7.65(m, 6H), 5.51 (d, 2H, J=13.5 Hz) 4.26 (s, 4H), 4.15 (s, 2H), 1.22 (s,3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 164.3 (d, J=3.5 Hz), 135.4 (d, J=3.1Hz), 133.9 (d, J=10.8 Hz), 130.4 (d, J=13.2 Hz), 117.4 (d, J=89.2 Hz),79.1, 71.1, 38.8, 33.6 (d, J=56.6 Hz), 21.0 ppm; ³¹P NMR (162 MHz,CDCl₃) δ 20.41 (s, 1P) ppm; HRMS (ES⁺) calcd for C₂₅H₂₆O₃P 405.1620found 405.1631.

9. To a flame-dried round bottom flask were added phosphonium iodide 8(831 mg, 1.56 mmol) and CH₂Cl₂ (10 mL), and a N₂ atmosphere wasestablished. DBU (0.212 mL, 1.42 mmol) was added, and the reactionmixture was stirred for 20 min. In a separate flame-dried round bottomflask, difluorodiketone 1 (250 mg, 1.42 mmol) was dissolved in CH₂Cl₂(20 mL), and this solution was then transferred to the first solution.The reaction mixture was allowed to stir for 24 h, concentrated underreduced pressure, and purified by flash chromatography (10:1hexanes:EtOAc), yielding a white solid (364 mg, 85%). R_(f)=0.50 (2:1hexanes:EtOAc); mp 44.2-46.0° C.; ¹H NMR (500 MHz, CDCl₃) δ 6.49 (s,1H), 4.51 (d, 2H, J=6.0 Hz), 4.40 (d, 2H, J=6.0 Hz), 4.25 (s, 2H), 2.81(t, 2H, J=6.6 Hz), 2.66 (t, 2H, J=6.7 Hz), 1.82 (m, 2H), 1.72 (m, 2H),1.52 (m, 2H), 1.35 (s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 200.2 (t,J=28.1 Hz), 165.1, 150.4 (t, J=20.0 Hz), 121.1 (t, J=9.6 Hz), 114.2 (t,J=254.6 Hz), 79.7, 69.3, 39.2, 37.5, 26.72, 26.66, 26.0, 25.5, 21.3 ppm;¹⁹F NMR (376 MHz, CD₃CN) δ −113.03 (s, 2F) ppm; HRMS (FAB) calcd forC₁₅H₂₀O₄F₂ [M+H]⁺ 303.1408 found 303.1404.

10. To a round bottom flask were added α,β-unsaturated ester 9 (804 mg,2.66 mmol) and MeOH (30 mL). The system was flushed with N₂ and acatalytic amount of Pd/C was added. The system was then again flushedwith N₂, followed by H₂, and the reaction mixture was stirred under a H₂atmosphere for 18 h. The system was then again flushed with N₂, and thereaction mixture was diluted with CH₂Cl₂ (30 mL) and filtered throughCelite to remove the catalyst. The filtrate was then concentrated underreduced pressure. The crude material was then dissolved in CH₂Cl₂ (17mL) and transferred via syringe to a new flame-dried round bottom flask,which was under a N₂ atmosphere and contained 4 Å molecular sieves. Themixture was then cooled to 0° C. with stirring. In a separateflame-dried conical flask was prepared a 0.20 M solution of BF₃.OEt₂(100 μL) in CH₂Cl₂ (3.9 mL), and a portion of this solution (1.0 mL,0.20 mmol) was added to the reaction mixture via syringe. The reactionmixture was then brought to rt and was stirred for an additional 20 hbefore being quenched with Et₃N (0.5 mL). The reaction mixture was thenconcentrated under reduced pressure and was purified by flashchromatography (20:1 hexanes:EtOAc with 1% Et₃N over deactivated silicagel) to yield a white solid (731 mg, 90% over two steps). R_(f)=0.70(2:1 hexanes:EtOAc); mp 80.3-83.3° C.; ¹H NMR (500 MHz, CDCl₃) δ 3.89(s, 6H), 2.65 (m, 1H), 2.62-2.46 (m, 2H), 2.21 (d, 1H, J=14.6 Hz),2.10-1.94 (m, 2H), 1.90 (br s, 1H), 1.60 (m, 2H), 1.56-1.42 (m, 2H),1.36 (m, 2H), 0.80 (s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 205.8 (dd,J=29.7, 25.7 Hz), 119.3 (dd, J=257.6, 250.2 Hz), 109.0, 72.8, 39.2 (t,J=21.2 Hz), 38.9, 34.6 (t, J=4.7 Hz), 30.5, 27.0, 26.3 (d, J=7.2 Hz),24.8 (d, J=2.7 Hz), 23.3, 14.7 ppm; ¹⁹F NMR (376 MHz, CD₃CN) δ −103.10(d, 1F, J=246.4 Hz), −124.20 (dm, 1F, J=248.97 Hz) ppm.

11. To a flame-dried round bottom flask under a N₂ atmosphere was addedTHF (30 mL) followed by KHMDS (2.66 mL, 1.33 mmol). The reaction mixturewas cooled to −78° C. with stirring, and ketone 10 (354 mg, 1.16 mmol)in THF (15 mL) was added dropwise via syringe over 15 min. The reactionwas stirred for an additional 3 h, then Tf₂NPh (457 mg, 1.28 mmol) inTHF (15 mL) was added via syringe, and the system was slowly brought tort with stirring over 19 h. The mixture was then quenched withdeactivated silica gel, concentrated under reduced pressure, andpurified by flash chromatography (5-6.5% EtOAc:hexanes with 1% Et₃N overdeactivated silica gel) to yield a white solid (407 mg, 80%). R_(f)=0.72(2:1 hexanes:EtOAc); mp 81.2-83.3° C.; ¹H NMR (400 MHz, CDCl₃) δ 6.05(t, 1H, J=9.6 Hz), 3.89 (s, 6H), 2.74-2.06 (m, 1H), 2.51-2.31 (m, 2H),2.20 (dd, 1H, J=14.5, 1.7 Hz), 2.00-1.87 (m, 1H), 1.72-1.49 (m, 6H),0.80 (s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 143.2 (t, J=30.2 Hz),126.9, 119.2 (apt t, J=246.5 Hz), 118.6 (q, J=320.0 Hz), 108.9, 72.8,40.9 (apt t, J=22.3 Hz), 35.1, 30.5, 27.2, 26.6, 22.6, 21.8, 14.6 ppm;¹⁹F NMR (376 MHz, CDCl₃) δ −74.52 (s, 3F), −93.80 (d, 1F, J=269.8 Hz),−104.75 (dm, 1F, J=278.0 Hz) ppm; HRMS (FAB) calcd for C₁₆H₂₁O₆F₅S[M+H]⁺ 437.1057 found 437.1050.

12. In a round bottom flask, vinyl triflate 11 (407 mg, 0.932 mmol) wasdissolved in toluene and subsequently concentrated under reducedpressure 4 times to remove trace moisture. The material was thendissolved in THF (20 mL), a N₂ atmosphere was established, and thesolution was cooled to −20° C. with stirring. In a separate flame-driedround bottom flask, a 0.20 M solution of LDA was made by addingn-butyllithium (1.12 mL, 2.80 mmol) to a solution of diisopropylamine(475 μL, 3.36 mmol) in THF (12.4 mL) stirring under a N₂ atmosphere at−78° C. A portion of the LDA solution (5.6 mL, 1.12 mmol) was addeddropwise via syringe to the first mixture over 1 h. The reaction mixturewas then brought to rt over 30 min, quenched with deactivated silicagel, concentrated under reduced pressure, and purified by flashchromatography (5-6.5% EtOAc:hexanes over deactivated silica gel) toyield a white solid (158.7 mg, 59%). R_(f)=0.73 (2:1 hexanes:EtOAc); mp80.1-86.0° C.; ¹H NMR (500 MHz, CDCl₃) δ 3.90 (s, 6H), 2.54 (apt dq, 1H,J=23.5, 9.1 Hz), 2.40-2.25 (m, 2H), 2.25-2.11 (m, 2H), 2.10-2.02 (m,2H), 1.81-1.71 (m, 1H), 1.65 (dd, 1H, J=14.6, 10.3 Hz), 1.50 (apt quint,1H, J=7.5 Hz), 1.33 (m, 1H), 0.81 (s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃)δ 120.0 (t, J=238.3 Hz), 109.8 (t, J=11.2 Hz), 85.4 (dd, 47.1, 41.8 Hz),72.8, 51.9 (t, J=23.8 Hz), 35.2 (dd, J=4.5, 1.6 Hz), 32.8 (d, J=4.8 Hz),32.7 (d, J=2.1 Hz), 30.5, 29.9, 28.2, 20.5, 14.8 ppm; ¹⁹F NMR (376 MHz,CD₃CN) δ −94.82 (d, 1F, J=258.7 Hz), −101.81 (ddt, 1F, J=259.3, 24.1,7.2 Hz) ppm.

13. To a scintillation vial were added difluorocyclooctyne orthoester 12(90.4 mg, 0.316 mmol), MeOH (4.5 mL), water (450 μL), and PPTS (159 mg,0.632 mmol). The reaction mixture was stirred at rt for 24 h, thenquenched with saturated NaHCO₃ (2 mL) and concentrated under reducedpressure. The material was then diluted with brine (8 mL) and extractedwith EtOAc (15 mL, 1×; 5 mL, 2×). The combined organic layer was thenwashed with brine (10 mL, 1×) and a HCl/brine solution (1:1 1 MHCl:brine, 10 mL, 2×), dried over MgSO₄, and filtered through a glassfrit. The filtrate was then concentrated under reduced pressure to yielda white solid. A portion of this material (57.8 mg) was transferred to around bottom flask, where it was dissolved in dioxane (1 mL) and water(200 μL). To this was added LiOH (91 mg, 3.8 mmol) and the reactionmixture was stirred at rt for 3 h, then quenched with 1 M HCl (5 mL).The mixture was further diluted with brine (3 mL) and extracted withEtOAc (10 mL, 4×). The combined organic layers were then washed with andHCl/brine solution (1:1 1 M HCl:brine, 10 mL), dried over MgSO₄, andfiltered through a glass frit. The filtrate was then concentrated andpurified by flash chromatography (20:1 hexanes:EtOAc with 2% AcOH) togive a white solid (33.2 mg, 86% over two steps). R_(f)=0.66 (1:1hexanes:EtOAc with 1% AcOH); mp 87.4-88.9° C.; ¹H NMR (400 MHz, CDCl₃) δ11.90-10.60 (br s, 1H), 2.84-2.69 (m, 2H), 2.46-2.28 (m, 3H), 2.21-2.05(m, 2H), 1.90-1.74 (m, 2H), 1.67 (m, 1H), 1.41 (m, 1H) ppm; ¹³C NMR (125MHz, CDCl₃) δ 177.8, 119.1 (t, J=238.6 Hz), 110.7 (t, J=11.2 Hz), 84.7(dd, J=47.0, 41.6 Hz), 52.6 (t, J=24.4 Hz), 33.7 (apt d, J=4.4 Hz), 32.9(d, J=4.6 Hz), 32.7 (d, J=2.0 Hz), 27.9, 20.5 ppm; ¹⁹F NMR (376 MHz,CDCl₃) δ −94.64 (d, 1F, J=260.0 Hz), −100.82 (ddt, 1F, J=260.2, 21.1,6.8 Hz) ppm; HRMS (ES⁻) calcd for C₁₀H₁₁O₂F₂ [M]⁻ 201.0722 found201.0729.

Kinetic evaluation of [3+2] cycloaddition of DIFO2 and DIFO3 with benzylazide. Stock solutions of DIFO2 (7, 20 mM) or DIFO3 (12, 20 mM) andbenzyl azide (200 mM) were made in CD₃CN. An NMR tube was charged with450 μL of the solution of 1, and, immediately before lowering into theNMR magnet, 50 μL of the benzyl azide solution, and the reaction wasmonitored over time using ¹H NMR spectroscopy. The kinetic data werederived by monitoring the change in integration of resonancescorresponding to the benzylic protons in benzyl azide (δ ˜4.4 ppm)compared to the corresponding resonances of the triazole products (δ˜5.5 to 5.7 ppm).

The second-order rate constant for the reaction was determined byplotting 1/[benzyl azide] versus time, followed by subsequent analysisby linear regression. The second-order rate constant (k, M⁻¹ s⁻¹)corresponds to the determined slope.

Cell surface labeling of azido glycans on Jurkat cells with biotinylatedconjugates—a comparison between DIFO-biotin, DIFO2-biotin, andDIFO3-biotin.

Jurkat (human T-cell lymphoma) and Chinese hamster ovary (CHO) cellswere maintained in a 5% CO₂, water-saturated atmosphere and grown inRPMI-1640 (Jurkat) or F12 (CHO) media supplemented with 10% FCS,penicillin (100 units/mL), and streptomycin (0.1 mg/mL). Cell densitieswere maintained between 1×10⁵ and 1.6×10⁶ cells/mL.

Jurkat cells were incubated for 1-3 d in untreated media or mediacontaining 25 μM Ac₄ManNAz. The cells were then distributed into a96-well V-bottom tissue culture plate, pelleted (3500 rpm, 3 min), andwashed twice with 200 μL of labeling buffer (PBS, pH 7.4 containing 1%FCS). Cells were then incubated with DIFO-biotin, DIFO2-biotin, orDIFO3-biotin in labeling buffer for 1 h at rt at various concentrations(1-10 μM) with dilutions made from a 2.5 mM stock in 7:3 PBS:DMF. Afterincubation, cells were pelleted, washed twice with labeling buffer, andresuspended in the same buffer containing fluorescein isothiocyanate(FITC)-avidin (1:200 dilution of the Sigma stock). After a 15-minincubation on ice (in the dark), the cells were washed once with 200 μL,incubated with FITC-avidin for an additional 15 min on ice, washed threetimes with 200 μL of cold labeling buffer, and then diluted to a volumeof 400 μL for flow cytometry analysis.

The data are presented in FIGS. 12 and 13. FIG. 12 presents comparativecell surface labeling of azido glycans by biotinylated derivatives ofDIFO, DIFO2, and DIFO3. FIG. 13 depicts concentration dependence of thereaction of DIFO2-biotin and DIFO3-biotine with cell surface azidoglycans. The x-axis is the concentration in μM; the y-axis is MFI, meanfluorescence intensity. Jurkat cells were incubated with 0 or 25 μMAc₄ManNAz for 3 days. (FIG. 12) The cells were then reacted with 10 μMDIFO-biotin, DIFO2-biotin, or DIFO3-biotin for 1 h (FIG. 12), or reactedwith various concentrations of DIFO2-biotin or DIFO3-biotin for 1 h(FIG. 13). In both cases, the cells were then stained with FITC-avidin,and analyzed by flow cytometry. Shown is the mean fluorescence intensity(MFI, arbitrary units). Error bars represent the standard deviation oftriplicate samples.

The data presented in FIGS. 12 and 13 show that DIFO2-biotin andDIFO3-biotin can selectively label azide-containing membrane-associatedglycans on live Jurkat cells; further, the data demonstrate that thesensitivity of azide detection using DIFO2 and DIFO3 is roughly 40% ofthat using DIFO.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A compound of the formula:

wherein Y is H; a moiety that comprises a reactive group thatfacilitates covalent attachment of a molecule of interest; or a moleculeof interest; wherein R₁ is selected from a carboxylic acid, an alkylester, an aryl ester, a substituted aryl ester, an aldehyde, an amide,an aryl amide, an alkyl halide, a thioester, a sulfonyl ester, an alkylketone, an aryl ketone, a substituted aryl ketone, a halosulfonyl, anitrile, and a nitro, wherein R₁ can be at any position on thecyclooctyne group other than at the two carbons linked by the triplebond, and other than the fluoride-substituted carbon.
 2. The compound ofclaim 1, wherein Y is a reactive group that facilitates covalentattachment of a molecule.
 3. The compound of claim 2, wherein thereactive group is selected from a carboxyl, an amine, an ester, athioester, a sulfonyl halide, an alcohol, a thiol, a succinimidyl ester,an isothiocyanate, an iodoacetamide, a maleimide, and a hydrazine. 4.The compound of claim 1, wherein Y is a molecule of interest selectedfrom a detectable label, a toxin, a linker, a peptide, a drug, a memberof a specific binding pair, and an epitope tag.
 5. The compound of claim1, wherein the compound is of the structure:


6. A compound of the structure:

wherein R₈ is selected from H; a halogen atom; an aliphatic group, asubstituted or unsubstituted alkyl group; an alkenyl group; an alkynylgroup; a carboxylic acid, an alkyl ester; an aryl ester; a substitutedaryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; analkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an arylketone; a substituted aryl ketone; a halosulfonyl; a nitrile; and anitro.
 7. A compound of the structure of one of Formulas XI-XVI:

wherein R, R₁, R₂, and R₃ are each independently H; a carboxylic acid; amethoxy; an alkyl ester; an aryl ester; a substituted aryl ester; analdehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; athioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; asubstituted aryl ketone; a halosulfonyl; —W—(CH₂)_(n)—Z (wherein n is aninteger from 1-4, wherein W, if present, is O, N, or S; and Z is nitro,cyano, sulfonic acid, or a halogen); —(CH₂)_(n)—W—(CH₂)_(m)—Z (wherein nand m are each independently 1 or 2; W is O, N, S, or sulfonyl, whereinif W is O, N, or S, then Z is nitro, cyano, or halogen, and wherein andif W is sulfonyl, then Z is H); or —(CH₂)_(n)—Z (wherein n is an integerfrom 1-4, and wherein Z is nitro, cyano, sulfonic acid, or a halogen);and; wherein each R, R₁, R₂, and R₃ is optionally independently linkedto a moiety that comprises a reactive group that facilitates covalentattachment of a molecule of interest; or a molecule of interest.
 8. Thecompound of claim 7, wherein: R and R₂ are independently selected from—COCH₂CH₂CO₂H and —CO-phenyl-CO₂H.
 9. The compound of claim 7, whereinthe compound is of one of the following structures: